U.S. patent application number 10/072509 was filed with the patent office on 2002-09-12 for method and apparatus for preparing fibrinogen adhesive from whole blood.
Invention is credited to Epstein, Gordon H..
Application Number | 20020128683 10/072509 |
Document ID | / |
Family ID | 23468141 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020128683 |
Kind Code |
A1 |
Epstein, Gordon H. |
September 12, 2002 |
Method and apparatus for preparing fibrinogen adhesive from whole
blood
Abstract
A process and apparatus for one-step preparation of fibrinogen
adhesive by polyethylene glycol-mediated precipitation from plasma
are disclosed. The methods and apparatus of the invention permit
preparation of autologous fibrinogen adhesive composition from the
patient during surgery, and can be applied generally to provide
such compositions. Also disclosed are an apparatus and method for
application of sealant comprising this fibrinogen adhesive
composition.
Inventors: |
Epstein, Gordon H.;
(Fremont, CA) |
Correspondence
Address: |
ATTN: Bruce M. Canter
OPPENHEIMER WOLFF & DONNELLY LLP
840 Newport Center Drive, Suite 700
Newport Beach
CA
92660
US
|
Family ID: |
23468141 |
Appl. No.: |
10/072509 |
Filed: |
February 5, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10072509 |
Feb 5, 2002 |
|
|
|
08863883 |
May 28, 1997 |
|
|
|
6394975 |
|
|
|
|
Current U.S.
Class: |
606/214 ;
604/82 |
Current CPC
Class: |
A61L 24/106 20130101;
A61B 17/00491 20130101; C07K 14/75 20130101; A61K 38/00 20130101;
A61L 24/0005 20130101; Y10S 514/802 20130101; A61B 2017/00495
20130101; Y10S 604/902 20130101; Y10S 435/81 20130101 |
Class at
Publication: |
606/214 ;
604/82 |
International
Class: |
A61D 001/00 |
Claims
I claim:
1. A process for preparing fibrinogen adhesive from whole blood
which process comprises: directly contacting plasma which has been
removed from said whole blood with an amount of polyethylene glycol
(MW 200-8000) effective to precipitate said fibrinogen; recovering
the precipitated fibrinogen; and suspending said precipitate in
aqueous medium.
2. The process of claim 1 wherein the plasma is prepared by
centrifugation of whole blood treated with anticoagulant.
3. The method of claim 2 wherein the anticoagulant is citrate
buffer.
4. The method of claim 1 wherein the PEG has a molecular weight of
1,000-1,500.
5. The method of claim 4 wherein said effective amount is 8-15%
w/v.
6. The method of claim 1 wherein the fibrinogen precipitate is
recovered by centrifugation.
7. A fibrinogen adhesive composition prepared by the method of
claim 1.
8. A pharmaceutical composition effective for cementing tissues
which comprises the fibrinogen adhesive composition of claim 7 in
admixture with thrombin and calcium ion.
9. A method to cement tissues which method comprises applying to
said tissues an effective amount of the fibrinogen adhesive
composition of claim 7 and an effective amount of thrombin and
calcium ion.
10. An apparatus for preparing a fibrinogen adhesive composition
from human blood, which apparatus comprises: a first chamber in
fluid communication with a second chamber, means for effecting said
fluid communication, and means for interrupting said fluid
communication; means for withdrawing a sample of human blood into
said first chamber in the presence of anticoagulant; said first
chamber being configured so as to permit separation of plasma and
red blood cells in said blood sample; a means to dispense said
separated plasma from said first chamber to said second chamber
free of said red blood cells wherein said second chamber contains
an amount of polyethylene glycol effective to precipitate
fibrinogen from said plasma, said second chamber being configured
so as to permit separation of the precipitated fibrinogen from the
remaining plasma; means to dispose of said remaining plasma.
11. The apparatus of claim 10 wherein said each of first and second
chambers is a body of a syringe.
12. The apparatus of claims 11 wherein said means for dispensing
plasma comprises a plunger fitted to the syringe body.
13. The apparatus of claim 12 wherein said means for interrupting
fluid communication includes a valve between said first and second
chambers.
14. The apparatus of claim 12 wherein said means for interrupting
fluid communication includes a means for detaching said means for
effecting fluid communication.
15. An applicator device for injecting a two-component system into
an animal subject, which applicator comprises a first and second
dispensing means each having an inlet end and an outlet end
disposed in parallel so as to permit the two components to be mixed
when dispensing pressure is applied to the first and second
dispensing means; a means for applying dispensing pressure to said
first and second dispensing means; and disposed adjacent to said
first and second outlet ends, a means to apply suction adjacent to
and at said outlet ends.
16. A method to apply sealant to a tissue to be sealed which
sealant comprises a fibrinogen adhesive component and a
Ca.sup.+2/thrombin component, which method comprises dispensing
said components into a premixing space adjacent to the tissue to be
sealed to form the sealant, and applying suction to the tissue to
be sealed to remove debris and excess sealant.
17. A method to apply sealant to a tissue to be sealed with sealant
comprises a fibrinogen adhesive component and a Ca.sup.+2/thrombin
component, which method comprises dispensing said components from
the apparatus of claim 15.
Description
TECHNICAL FIELD
[0001] The invention is related to medical and veterinary
technology and procedures. More specifically, it concerns a
convenient method for preparation of a fibrinogen adhesive directly
from withdrawn blood.
BACKGROUND ART
[0002] The need for a suitable tissue adhesive for use in surgical
procedures in human and animal medicine is well understood, and has
been for nearly a century. Various substances have been tried,
including completely synthetic materials such as cyanoacrylate.
Because, however, the ability of the natural material fibrin to
behave as an adhesive is known, fibrin powders were used for this
purpose early in the development of this technology.
[0003] It has also long been recognized that the in situ formation
of fibrin from fibrinogen is a preferable approach. In a relatively
recent article, Staindl, O., in Ann. Otol. (1979) 88:413-418,
described the use of a combination of three separately prepared
substances, human fibrinogen cryoprecipitate, thrombin in the
presence of calcium ion, and Factor XIII concentrate, to obtain a
glue that was applied in skin graft applications, myringoplasty,
repair of dural defects, hemeostatis after tonsillectomy, and
tracheoplasty. Around this time, Immuno-AG, Vienna, Austria, began
producing and commercializing a two-component "fibrin seal" system,
wherein one component contains highly concentrated human
fibrinogen, Factor XIII, and other human plasma proteins, prepared
from pooled blood, and the other component supplies thrombin and
calcium ion. The two components are added together in the presence
of a fibrinolysis inhibitor. After application, the processes of
coagulation and fibrin cross-linking occur. Eventually, the seal
may lyse in the process of healing of the wound or trauma which
accompanies the reconstruction of the tissue. Redl, H., et al.,
"Biomaterials 1980," Winter, G. D., et al., eds. (1982), John Wiley
& Sons, Ltd., at page 669-675, describe the development of an
applicator device for this system which mixes and applies the two
components of the system simultaneously.
[0004] This two-component system has been marketed over the last
ten years under the trade name Tissucol.TM. or Tisseel.TM., and a
number of publications have appeared describing the use of this
preparation in surgical procedures. See, for example, Seelich, T.,
J Head and Neck Pathol. (1982) 3:65-69; O'Connor, A. F., et al.,
Otolaryngol Head Neck Surg. (1982) 90:347-348; Marquet, J., J Head
and Neck Pathol. (1982) 3:71-72; Thorson, G. K., et al., J Surg.
Oncol. (1983) 24:221-223. McCarthy, P. M., et al., Mayo Clin. Pros.
(1987) 62:317-319, reported the addition of barium ion to this
fibrin glue system in the treatment of a bleeding duodenal sinus in
order to facilitate follow-up surveillance. See also Portman, M., J
Head and Neck Pathol. (1982) 3:96; Panis, R., ibid., 94-95.
[0005] It has been recognized, of course, that the use of the
commercial product, which is prepared from pooled human blood,
poses at least a theoretical risk of contamination by
disease-producing organisms, especially viruses. The earlier
concerns were directed to transmission of hepatitis B, but more
recently the focus has shifted to concern about inadvertent
transfer of the AIDS virus. Therefore, attention has been given to
methods to isolate an autologous counterpart of the
fibrinogen-containing component in the Tissucol.TM.. system in a
practical manner for use in surgery. For example, Siedentop, K. H.,
et al., Laryngoscope (1985) 95:1074-1076, describes a number of
approaches to the precipitation of fibrinogen from plasma in the
context of the proposed use of this material as the
fibrinogen-furnishing component of a fibrin glue. Four methods were
suggested: Precipitation with ethanol, use of unfractionated
plasma, cryoprecipitation, and precipitation with ammonium sulfate.
The first three methods were considered by the authors to be
unsatisfactory; the fourth, which was the method described by
Harker, L. A., et al., New Eng J Med (1972) 287:999-1005, was
considered to have some promise.
[0006] Epstein, G. H., et al., Anals Otol Rhinol and Laryngol
(1986) 95:4045, suggest the use of a fibrinogen preparation from
autologous plasma obtained using polyethylene glycol precipitation.
These authors adapted the method of Masri, M. A., et al., Thromb
Hemeostas (1983) 49:116-119, which involved incubation of the
harvested plasma with barium sulfate and magnesium sulfate in order
to remove thrombin (and thus prevent premature fibrin formation)
followed by treatment of the supernatant from the resulting
precipitation with a 30% solution of polyethylene glycol (PEG) of
MW approximately 1000. This purified fraction was then mixed with
thrombin containing calcium ion in order to form the glue. The
isolation process required approximately two-three hours, but the
PEG-precipitated fraction could be stored for as much as several
weeks, and therefore the preparation could be made in advance of
surgery. Autologous fibrinogen preparations obtained following this
procedure were shown to be adhesive with implant materials by
Feldman, M. C., et al., Arch Otolaryngol--Head and Neck Surg (1988)
114:182-185; additional applications of this approach to other
surgical indications were also reported by Feldman, M. C., et al.,
Arch Ophthalmol (1987) 105:963-967; Feldman, M. C., et al., M J
Otolog (1988) 9:302-305; Silberstein, L. E., et al., Transfusion
(1988) 28:319-321.
[0007] In addition, the material prepared as described by Epstein
et al., by barium sulfate/magnesium sulfate precipitation followed
by treatment of the supernatant with polyethylene glycol, was
characterized to as to its components by Weisman, R. A., et al.,
Laryngoscope (1987) 97:1186-1190. The product was found to contain
about 13-50 mg/ml of fibrinogen when the material precipitated from
50 ml blood was brought to a volume of 1 ml, wherein this
concentration varies with the concentration of PEG. The percent of
total protein attributable to fibrinogen ranged from 42-92 percent;
the lower the PEG concentration, the higher the percentage of
fibrinogen. The concentrations of kallikrein and pre-kallikrein
were shown to be reduced to 1-2% of their levels in plasma in the
presence of 10% PEG, and Factor XIII activity was shown to be
present.
[0008] A system for preparing autologous tissue adhesive was also
described by WeisFogh in PCT Application ______, which discloses a
reasonably complex system based on ethanol precipitation.
[0009] General methods for preparing plasma adhesives have also
been described in a series of related U.S. patents by Schwarz et
al. assigned to Immuno-AG: U.S. Pat. Nos. 4,414,976; 4,298,598;
4,362,567; and 4,377,572. These methods involve cryoprecipitation
of the adhesive material from plasma and various work-up procedures
to obtain a mixture of defined composition. In addition, U.S. Pat.
No. 4,427,650 to Stroetmann describes a preparation method
involving the formation of a cryoprecipitate.
[0010] Very early studies on blood fibrinogen, unrelated to the
preparation of adhesive compositions, experimented with the use of
glycine as a precipitant. A 1963 paper by Kazal, L. A., et al.,
Proc Sec Exp Biol Med (1963) 10:989-994, described the use of
glycine at about 2.2M at room temperature to precipitate fibrinogen
after removal of the thrombin with BaSO.sub.4/MgSO.sub.4
precipitation. Moesson, M. W., et al., Biochemistry (1966)
5:2829-2835, recognized that fibrinogen can be directly
precipitated in this manner, but did not assess the suitability of
the precipitated material to behave as a controlled adhesive.
[0011] None of the foregoing methods is readily adaptable for
convenient use of an autologous plasma fraction as an adhesive
which can be prepared quickly in the course of the surgical
procedure. All of the approaches suggested for preparation of the
fibrinogen-containing fraction for this purpose are too
time-consuming and complex to be finished in a short enough time
period to be accomplished during the course of the surgery. Also,
in some procedures, such as cryoprecipitation, special equipment,
such as refrigerated centrifuges, is required. While the prior art
approach is to prepare the composition in advance, this immediately
imposes the necessity for additional procedures for identification
and retrieval of the samples matched with the patient, and the
concomitant opportunity for error, in addition to the inconvenience
to the patient, who must then arrange time for an additional
medical appointment. And, of course, this practice is not possible
when the surgery is conducted on an emergency basis.
[0012] The present invention, by providing a surprisingly simple
method and apparatus for the preparation of autologous adhesive,
permits the autologous composition to be prepared in the operating
room and administered at the time of preparation.
[0013] In addition to the problem of preparation, methods to
administer the adhesive have not provided sufficient controls to
permit clean fusions of tissue to be made. The adhesive is
administered as a two-component system--the fibrinogen preparation
as one component and a thrombin/Ca.sup.+2 composition as another.
The Tissucol.TM. system uses a double-syringe arrangement wherein
the two components are mixed in the injector immediately prior to
dispensing. No means to remove debris or excess adhesive from the
site of application is provided, and the potential for premature
adhesion also exists. The present invention also provides an
applicator with a premix immediately upstream of the application
outlet means and a means to apply suction at the outlet to maintain
the application area in condition for successful adhesion.
DISCLOSURE OF THE INVENTION
[0014] The invention provides effective means to prepare and
administer autologous plasma-derived adhesive during the course of
a surgical procedure. The method of the invention can also be used
to prepare the adhesive under less constrained conditions, but the
simplicity and ease with which the invention method results in the
desired adhesive makes it adaptable to this important use.
[0015] The invention process provides the desired adhesive
composition in a one-step treatment directly applicable to
separated plasma using a recognized, nontoxic precipitant, such as
suitable concentrations of amino acids, but preferably a nontoxic
polymer such as polyvinyl alcohol (PVA) or polyethylene glycol
(PEG). The fraction precipitated by these pharmaceutically
acceptable reagents results in a high concentration of fibrinogen
in the precipitate, along with other factors necessary for fibrin
formation, as well as permitting the use of a simple, disposable
apparatus to carry out the manipulations.
[0016] Thus, in one aspect, the invention is directed to a process
for preparing a fibrinogen adhesive composition from whole blood,
which process comprises directly contacting plasma with an amount
of polyethylene glycol of molecular weight 200-8000, or other
precipitant selected from the group consisting of amino acids and
nontoxic hydrophilic polymers, which is effective to precipitate
the fibrinogen-containing fraction. Then the precipitated
fibrinogen fraction is recovered for use, either by mechanical
disruption of the precipitate or pellet or by suspension in an
aqueous medium.
[0017] In another aspect, the invention is directed to the
fibrinogen-containing preparation obtained by the method of the
invention.
[0018] In still another aspect, the invention is directed to an
apparatus for preparing the fibrinogen composition consisting
essentially of a device with dual separation chambers with a means
for fluid communication between the chambers.
[0019] In still another aspect, the invention is directed to an
applicator for injecting this or any other two-component system
into a human or other animal subject which provides for
simultaneous application of suction at the outlet for the
components, and to a method to administer the fibrinogen
composition using this apparatus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a perspective view of one embodiment of the
apparatus of the invention for preparing the plasma-derived
adhesive.
[0021] FIG. 2 is an alternative embodiment of the isolation
apparatus of the invention.
[0022] FIG. 3 is an additional alternative embodiment of the
isolation apparatus of the invention.
[0023] FIG. 4 is a longitudinal sectional view of an apparatus for
administering a two-component composition to an animal subject.
[0024] FIG. 5 is a perspective view of the apparatus of FIG. 3.
[0025] FIGS. 6A and 6B are perspective views of the mixing head of
the apparatus of FIGS. 4 and 5.
[0026] FIG. 7 is a perspective view of the filler tray and
reservoir tip of the apparatus of FIGS. 4 and 5.
MODES OF CARRYING OUT THE INVENTION
[0027] The Adhesive Composition
[0028] The adhesive composition of the invention contains
substantial amounts of fibrinogen and sufficient Factor XIII to
effect fibrin formation and cross-linking in the presence of added
thrombin and calcium ion. Thus, as used herein, "fibrinogen
adhesive composition" refers to a fraction of blood plasma which
contains substantial amounts of fibrinogen and sufficient amounts
of other factors required for fibrin formation that, when combined
with a preparation of thrombin and calcium ion, fibrin formation
and cross-linking takes place conferring on the composition the
properties of a tissue adhesive.
[0029] It is believed that the formation of the adhesive occurs
through the conversion of fibrinogen into fibrin monomers, which
conversion is mediated by thrombin; these monomers aggregate and
form a gel. Thrombin also catalyzes the transformation of Factor
XIII to Factor XIII* which is activated in the presence of calcium
ions to Factor XIIIa. Factor XIIIa cross-links the fibrin monomers
of the gel aggregation into a polymeric state. The polymeric
material adheres to tissue or bone, and continued cross-linking can
occur as Factor XIIIa also cross-links fibronectin.
[0030] Therefore, the necessary components of the fibrinogen
adhesive composition are fibrinogen itself and Factor XIII. Other
components may also be present or may be added to modify and
improve the effect of the adhesive. For example, a fibrinolysis
inhibitor, such as aprotinin or aminocaproic acid can also be
added. In the compositions of the invention, the levels of
kallikrein and prekallikrein are greatly reduced from those present
in plasma; these factors are also fibrinolytic. Other additives
useful for purposes not directly related to the adhesive properties
of the composition may also be included, such as various protein
factors which are useful in wound healing, for example, fibroblast
growth factor, platelet-derived growth factor, various angiogenic
factors and the like. Other hormones, such as insulin, glucagon,
steroids, and prostaglandins can also be included, resulting in a
slow release system for these drugs. Also because the invention
compositions may be prepared sufficiently efficiently to permit the
use of an autologous fraction, other factors from plasma which aid
in clotting, such as Factor VIII and immunoglobulins, are
beneficial rather than detrimental when brought down with the
fibrinogen precipitate. By adjusting the concentration of the
precipitant and/or, in this case of a polymeric precipitant, its
molecular weight, variable amounts of these factors may be included
in the composition.
[0031] Preparation of the Composition
[0032] The method of the invention which provides the fibrinogen
adhesive composition is straightforward and sufficiently simple
that it can be used in the operating room to provide an autologous
adhesive preparation. The appropriateness of the process for this
application does not mean, of course, that its application is thus
limited. This straightforward process can be used as well in the
preparation of fibrinogen adhesive compositions in advance of
surgery from autologous, designated donor, or pooled plasma
sources, if desired. Furthermore, while its use is envisioned as
most dramatically associated with surgery in humans, it is also
suitable for veterinary uses.
[0033] The process results in a composition which has 50-80 mg/ml
of fibrinogen, in general, as compared to the 2-6 mg/ml
concentration in plasma or whole blood. This is a range comparable
to the commercially available Tisseel.TM. composition which
contains 70-110 mg/ml. Also, like the commercial Tisseel.TM.
composition, the fibrinogen adhesives of the present invention
contain sufficient Factor XIII to provide cross-linking of the
fibrin obtained. There may also be present varying amounts of
plasma components which are not absolutely necessary for fibrin
formation but may be helpful, such as, e.g., Factor VIII,
prostaglandins, and platelets.
[0034] In general, the process begins with the withdrawal of whole
blood from an autologous, or other, or multiple donors in the
presence of an anticoagulant, and separation of plasma from the red
blood cell fraction. Typical anticoagulants include heparin and
sodium citrate; others generally known in the art may also be used.
If sodium citrate is used, it is generally supplied as a buffer at
physiological pH with citrate ion present at 3-5%. The sodium salt
is preferred. The range of means to prepare citrate and other
anticoagulants suitable for primary separation of blood into red
blood cells and plasma is well understood in the art. Typically,
the separation can be effected by centrifugation; a suitable
protocol involves spinning at about 2000-5000 g for about 5-10
minutes.
[0035] In the method of the invention, after the plasma is
separated from the red blood cells, it is treated directly, at
ambient temperature, without prior treatment to remove thrombin,
with a physiologically acceptable nontoxic precipitant. The
precipitants of the invention include the natural amino acids and
nontoxic polymers, especially polyvinyl alcohol and, most
preferably, polyethylene glycol. Preferred amino acids are small
neutral amino acids such as glycine, serine and alanine, most
preferably glycine. Most preferred among the polymer precipitants
is polyethylene glycol in a molecular range of 200-8000 supplied as
a concentrated solution. The precipitant is preferably supplied as
a concentrated solution which, when mixed with plasma, will yield a
concentration effective in precipitating the adhesive composition.
Optimal concentration of the precipitant can readily be determined
by routine experimentation; simple in vitro assays for fibrin
formation, such as those described by Epstein, G. H., et al., Anals
Otol Rhinol and Laryngol (1986) 287:999-1005, can be used, and
fibrinogen content measured as described.
[0036] The concentrated solution or, in some instances, undiluted
precipitant is added to the plasma in an amount effective to
precipitate the fibrinogen adhesive composition of the invention.
If PEG is used, the final concentration of PEG depends on the
concentration of plasma in the precipitating solution and on the
molecular weight of the PEG. A suitable and typical set of
parameters utilizes an approximately 60% solution of PEG of
molecular weight 1000 in saline at pH 7.4, and the plasma is added
in sufficient volume (typically 10% w/v of precipitant
solution/final volume) to obtain a resulting concentration of 8-15%
w/v PEG in the volume of the final mixture. The plasma itself is,
therefore, only slightly diluted. The precipitation takes place at
room temperature and is complete essentially instantaneously,
although generally it is advantageous to cure the precipitate for
5-10 min.
[0037] The precipitate containing the fibrinogen adhesive is then
recovered, typically by centrifugation. The pellet may be recovered
by mechanical disruption or resuspended in normal saline for use at
a concentration of about 50 mg/ml-80 mg/ml of fibrinogen. Typically
from about 60 cc of whole blood a fibrinogen adhesive composition
which would contain 30-50 mg fibrinogen when resuspended to 1.5 ml
is obtained.
[0038] The use of the amino acids and biocompatible polymers as
precipitant has considerable advantage over the prior art use of
ethanol and cryoprecipitation. The resulting concentration of
fibrinogen is markedly higher, and the precipitants are recognized
material safe for internal use. For example, PEG has been used as a
lubricant or conditioner in ointments and in pharmaceutical and
cosmetic preparations, including injectables. It is a
water-soluble, nonvolatile material of no known clinical toxicity.
The amino acids are, of course, nontoxic metabolites and are common
components of pharmaceutical compositions.
[0039] Because of the benign nature of the precipitant, the
remaining supernatant plasma fraction, which contains platelets and
white blood cells, can be reused for other purposes; similarly, the
red blood cells removed could, if desired, be returned to the
patient.
[0040] Isolation Apparatus
[0041] The process of the invention can be conducted using standard
laboratory and medical equipment. For example, the blood may simply
be withdrawn into a syringe containing anticoagulant and emptied
into a centrifuge tube for separation. The supernatant plasma can
then be removed by a syringe containing the precipitant or can be
pipetted into a separate vessel and the precipitant added. The
suspension can then be centrifuged using standard equipment and the
precipitated material disrupted or resuspended by addition of
saline after removal of the supernatant.
[0042] It is evident, however, that the use of standard equipment
adds complexity to the process and imposes requirements for
sterilization of the various pieces of apparatus and maintenance of
integrity of the sample. The invention apparatus provides a simple
design which eliminates these complexities and streamlines the
process.
[0043] The invention apparatus contains two chambers, both of which
can be used directly in a laboratory or medical centrifuge. The
first chamber is designed to effect the separation of plasma and
red blood cells; the second chamber provides a method to separate
the fibrinogen adhesive composition from the supernatant plasma
fraction. Because a means of fluid communication is provided
between the first and second chamber, the plasma obtained in the
first chamber can be made the subject of fibrinogen adhesive
separation in the second, and the supernatant from the precipitate
can be returned to the first chamber. The first chamber generally
contains an effective amount of anticoagulant; the second chamber
contains an effective amount of precipitant such as polyethylene
glycol solution to effect precipitation of the fibrinogen adhesive.
The apparatus, then, comprises a first and second chamber, a means
of fluid communication between the first and second chambers and a
means to interrupt this communication, wherein the first chamber
contains anticoagulant and the second chamber contains the
precipitant and permits the removal of the precipitate.
[0044] One convenient embodiment of the apparatus of the invention
is shown in FIG. 1. In this embodiment, the apparatus consists of
two especially adapted syringe bodies 10 and 11 interconnected by a
supported tube 12. The tube is provided with a valve 13 which is
capable of turning on and off the flow of fluid between chambers 10
and 11. Both of the chambers are fitted onto the frame and locked
onto a tube having a T-joint at 10t and 11t. Both syringe cylinders
are provided with a modified plunger 10p and 11p which may be
withdrawn by a pulling means such as a plastic cord 10c and 11c
causing the withdrawal of the plunger to a position flush with the
ends of the cylinders where the top of the plunger is sealed by a
flange (10f and 11f) when withdrawn to a fully extended position.
This permits the syringe bodies to be used as centrifuge tubes when
the plunger is fully withdrawn to the end of the syringe and sealed
at the flange. While the plunger could be returned to the position
toward the tip of the syringe by use of a rod inserted at the
apertures 10a and 11a, this is generally not necessary as the
material in the chamber can be removed by suction, provided either
by the adjacent chamber or by the application of a vacuum at the
chamber outlet. Thus, withdrawal of the plunger in the chamber 11
with valve 13 open and the outlets 10o and 11o sealed results in
transfer of the liquid from chamber 10 to chamber 11. Both syringes
can be detached from the supporting frame 14 by release of the
locks to the T-tube, and can be sealed if appropriate.
[0045] In use, the plasma separation chamber 10 is fitted with a
needle 10n which is inserted into the subject for withdrawal of
blood. The valve 13 is closed during this step, thus preventing the
formation of a vacuum in the connecting tube 12 and confining the
flow of blood to the chamber 10 when the plunger 10p is withdrawn
by pulling the cord 10c. The size of the plunger is adjusted to
permit withdrawal of a predetermined amount of blood, typically 60
cc. The chamber 10 is previously supplied with sufficient citrate
buffer to obtain a concentration of about 4% citrate ion when the
predetermined amount of blood is withdrawn.
[0046] When the blood is withdrawn into chamber 10, the needle is
detached, the outlet 10o sealed with a sealing cap, and the device
removed to a centrifuge with a head designed to accommodate this
device, or, if desired, chamber 10 is detached for centrifugation
independently. The application of 4500 g for 5-10 minutes results
in a separation of the red blood cells from the plasma. If the
device is placed intact into the centrifuge, the plunger of chamber
11 is unaffected as the outlet of 11, shown as 11o, is sealed and
the-PEG contained in this chamber is not compressible.
[0047] After centrifugation, the valve 13 is opened and the plunger
11p is withdrawn and sealed at flange 11f, thus transferring the
plasma into chamber 11. Chamber 11 is presupplied with an
approximately 60% PEG solution of molecular weight 1000, or a
comparable concentration of an alternate molecular weight
sufficient to effect precipitation of the fibrinogen adhesive. The
motion of the plungers is stopped when the red blood cells reach
the upper portion of the chamber 10. The valve 13 is then
closed.
[0048] If desired, the red blood cells remaining in chamber 10 can
be withdrawn or expelled and returned to the patient.
[0049] The apparatus is then agitated, if necessary, to intermix
the PEG with the plasma. Outlet 11o remains sealed. The chamber 11
is then centrifuged either within the device or after removal
therefrom to separate the precipitate from the supernatant plasma.
The apparatus is, of course, inverted from the position shown in
FIG. 1. After about 15-20 minutes at 6000-10,000 g sufficient
separation of precipitate has occurred. The chamber 11 is replaced
in the apparatus if it has been removed, and the valve 13 again
opened while the outlet 11o remains sealed. The supernatant is then
withdrawn into chamber 10 by withdrawal of plunger 10p (10o remains
closed) until the fibrinogen precipitate begins to enter the
connecting tube 12; valve 13 is then closed.
[0050] The fibrinogen adhesive is removed for use from chamber 11
by further reinsertion of the plunger 11p, or preferably by
withdrawal into the syringe attached at the outlet 11o. If desired,
the fibrinogen adhesive can be diluted in saline and the
composition quantitatively removed from chamber 11 by washing.
[0051] It should be noted that chamber 10 now contains all blood
components except the fibrinogen adhesive composition and contains
as added components only anticoagulant and the clearly nontoxic
PEG. Therefore, the modified blood in chamber 10 could, if
necessary, be used for other purposes or returned to the
patient.
[0052] FIG. 2 shows an alternative embodiment of the apparatus of
the invention which dispenses with the need for the frame 14. This
apparatus consists essentially of two syringes with detachable
plungers and a Luer-locked connector. This embodiment has the
advantage of utilizing standard medical equipment but the
disadvantage of utilization of several separate parts.
[0053] In the apparatus of FIG. 2, the blood separation syringe 20,
is fitted with a needle for the withdrawal of blood. Again, chamber
20 is supplied with a premeasured quantity of citrate which, when
mixed with the withdrawn blood will result in a citrate buffer of
about 4% citrate ion. When the plunger is retracted and chamber 20
contains whole blood, the needle and the extended portion of the
plunger are detached to permit the syringe body and sealed end to
be placed in a centrifuge, with the Luer lock outlet 20o sealed.
Suitable syringes with plungers which can be detached and the body
sealed for centrifugation of the syringe body are available
commercially. The syringe barrel 20 is then centrifuged as
described above, and Luer lock at 20o is engaged by the
interconnecting tube 24, and the plunger is replaced. With the
syringe in the inverted position, the interconnector 24 is attached
to a second similarly constructed syringe 21 at Luer lock 21o.
Syringe 21 contains a premeasured quantity of PEG. Plasma is
transferred from chamber 20 to chamber 21 through retraction of the
plunger 21p which causes effective reinsertion of plunger 20p. When
red blood cells begin to enter the interconnector, an event which
is readily observable, the interconnector 24 is detached from
chamber 21 and the mixture of plasma and PEG in chamber 21 agitated
if necessary. Syringe 21 is then prepared for centrifugation by
removal of the extended portion of the plunger and sealing of the
body of the syringe, and closure at 21o, and the fibrinogen
adhesive and plasma are separated under the centrifugation
conditions described above. The interconnector 24 and plunger 21p
are then put back in place and the plasma supernatant withdrawn
into chamber 20 by retraction of plunger 20p. When fibrinogen
adhesive precipitate begins to enter the interconnector, the
interconnector is detached and the fibrinogen adhesive expelled
into diluent, or diluent is withdrawn into chamber 21 through
21o.
[0054] In either case, the resuspended fibrinogen adhesive is
placed in a suitable applicator for use.
[0055] FIG. 3 shows a third embodiment of the apparatus for
isolation of the fibrinogen adhesive. Chambers 30 and 31 are
connected through the tubing 32, which is regulated by a valve 33.
Any suitable type of valve can be used; as shown in FIG. 3, valve
33 operates by sliding the roller so as to pinch off the tubing
32.
[0056] Chambers 30 and 31 are attached to each other by sealing or
by forming the chambers using the same mold; the chambers are, as
before, preloaded with suitable amounts of anticoagulant (chamber
30) and precipitant (chamber 31). Chambers 30 and 31 are provided
with outlets 30o and 31o at the sides of the chambers, as shown.
Outlet 30o is a Luer lock designed to accommodate a needle as
ordinarily attached to a syringe. Outlet 31 is a Luer lock designed
to accommodate an externally attached syringe body. Both outlets 30
and 31 have detachable sealing caps 30s and 31s.
[0057] The chambers 30 and 31 have plungers 30p and 31p which are
sealed with gaskets 30g and 31g to the sides of the chambers. When
fully withdrawn, the plungers are snug against the end plates 30e
and 31e at the bases of the chambers. The plungers are withdrawn by
a flexible plastic cord 30c and 31c, which are provided with disks
30d and 31d to aid in the withdrawal. Additional apertures 30a and
31a permit insertion of rigid rods to push the plungers forward, if
required; however, the movement of the plungers is generally
effected by application of a vacuum either by withdrawal of the
plunger in the adjacent chamber or by an externally attached
syringe.
[0058] The operation of the apparatus as shown in FIG. 3 is
substantially analogous to that shown in FIG. 1. In a typical
procedure, the patient's blood, about 60 cc, is withdrawn into
chamber 30 with valve 33 and outlet 31o both closed. Outlet 30o is
then sealed, the apparatus is placed in a centrifuge, and the
plasma and red blood cells in chamber 30 are separated. Valve 33 is
then opened and plunger 31p is withdrawn until the red blood cells
are seen entering the tubing 32. Valve 33 is closed, and the
apparatus is agitated, if necessary, and inverted and centrifuged
to separate the fibrinogen adhesives from the supernatant plasma.
The red blood cells in chamber 30 may be returned to the patient by
an attached needle and pushing the plunger 30p prior to
centrifugation, if desired.
[0059] After centrifugation, the valve 33 is again opened and
plunger 30p withdrawn to draw the supernatant into chamber 30.
Withdrawal of the plunger is stopped as soon as the supernatant has
left chamber 31.
[0060] Outlet 31o is opened and a syringe body attached. The
fibrinogen adhesive pellet is disrupted, for example, by repeated
motion of the syringe plunger, and, if desired, saline may be
included in the attached syringe to achieve quantitative transfer
of the fibrinogen adhesive. The adhesive composition is then placed
in a suitable applicator for administration to the subject.
[0061] Administration and Use
[0062] The fibrinogen adhesive composition of the invention is
useful in medical and veterinary applications generally comparable
to those known in the art for the commercially available product
Tissucol.TM.. Commercial preparations of thrombin and calcium ion
for admixture with the fibrinogen adhesive of the invention are
commercially available and are used as is generally known in the
art.
[0063] In a particularly advantageous way to apply the two
components of the sealant to the tissue to be cemented, a
dual-barreled applicator with one barrel for supply of the
fibrinogen adhesive and the other for the thrombin/calcium ion
mixture is employed wherein the outlet of each barrel is proximal
to a suction device so that the surface prior to application can be
instantly cleaned and excess components immediately removed.
[0064] Although the premixing of the components for this purpose is
known in the art, as described in U.S. Pat. No. 4,359,049,
incorporated herein by reference, the advantages of simultaneous
application of suction for controlling the environment of the
application have not been recognized. To applicants' knowledge, the
only medical devices which simultaneously permit application of a
material and suction are devices designed for irrigation, such as
that described in U.S. Pat. No. 4,299,221, where a fluid is applied
and then removed from a target tissue, especially in dental
applications, for cleansing purposes.
[0065] In the present application, the provision of suction at the
site of sealant application permits the withdrawal of interfering
substances as well as control of the quantity of sealant applied.
Thus, although the commercially available Duploject device can be
used for application, the modified adhesive applicator described
hereinbelow is highly advantageous. This device is designed to
permit controlled application of both fibrinogen adhesive and
thrombin/calcium mixture with premixing of these components
immediately prior to injection along with a means to produce
suction for surface cleaning and excess sealant removal.
[0066] A particularly advantageous design of such an applicator is
shown in FIGS. 4-7 herein, and was described in U.S. Ser. No.
06/683,621 filed Dec. 19, 1984 by the applicant, and now
abandoned.
[0067] In more detail, FIGS. 4 and 5 show a housing 2 which
resembles a pen in both size and shape, and which is held in a
similar fashion. The interior of the housing 2 includes a chamber 4
having walls that are sufficiently flexible so that the chamber 4
may be compressed by a relatively slight amount of external
pressure. At the same time, the walls of the chamber 4 are
sufficiently resilient so that, in the absence of external
pressure, the chamber 4 will return to its original volume and
configuration. The chamber 4 is divided into a first compartment 6
and a separate but identical second compartment 8. In application
of the composition of the invention to a subject, one chamber (6)
contains the fibrinogen adhesive composition and the other (8)
contains the thrombin/Ca.sup.+2 preparation. In the preferred
embodiment of the present invention, the chamber 4 consists of a
rubber bulb, although it is recognized that any chamber whose
volume may be easily reduced and expanded may serve as the chamber
4.
[0068] The first compartment 6 of the chamber 4 is connected to a
first liquid channel 10 which runs lengthwise through the interior
of the housing 2 and which terminates at the housing end piece 12.
The second compartment 8 of the chamber 4 is likewise connected to
a second liquid channel 14 which runs parallel to the first liquid
channel 91 and which similarly terminates at the housing end piece
92.
[0069] The housing end piece 92 is fitted with an adaptor 22 which
is configured to receive a connector 24 or 93 of a detachable
channel extension 25. The channel extension 25 includes two liquid
channels 26 and 28 (and a suction channel 97 to be described
below). When the channel extension 25 is attached to the housing
end piece 92 by means of the adaptor 22 and the connector 24 (FIG.
5) or 93 (FIG. 4), the liquid channels 28 and 26 serve as
extensions, respectively, of the fluid channels 91 and 95 and as
reservoirs for the fibrinogen adhesive and the Ca.sup.+2/thrombin
preparation.
[0070] The channel extension 25 may be configured in a variety of
lengths and shapes, depending upon the particular requirements of
the applicator. The use of a detachable channel extension 25 allows
a single applicator housing 2 to be used in conjunction with a
variety of differently configured channel extensions 25.
[0071] The present invention also includes a mixing head 94 (see
FIGS. 6A and 6B) The mixing head 94 includes three entry ports of
which two, 34 and 36, are configured to be attached respectively to
the distal ends 40 and 42 of the liquid channels 26 and 28.
[0072] The interior of the mixing head 94 includes a Y-shaped
channel 46 which connects the entry ports 34 and 36 with the exit
port 48. The liquid adhesive components dispensed through the entry
ports 34 and 36 are thereby combined in the channel 46 before being
ejected through the single exit port 48. A single adhesive channel
tube 50 is connected to the exit port 48, thus permitting the
premixed adhesive to be applied.
[0073] The apparatus as shown in FIGS. 4 and 5 includes a suction
channel 16 which is incorporated into the wall of the housing 2 and
which runs parallel to the first and second liquid channels 91 and
95. Suction channel 16 also terminates at the housing end piece 92.
The proximal end of the suction channel 16 is surrounded by an
adaptor 18 which is configured to connect the suction channel 16 to
a remote vacuum source (not shown).
[0074] Operation of the suction channel 16 is controlled by means
of a venturi opening 96 which is located on the exterior of the
housing 2. Obstruction of the venturi opening 96 permits the
suction force which originates at the remote vacuum source to be
conducted through the length of the section channel 16.
[0075] The interior of the mixing head 94, shown in FIGS. 6A and
6B, also includes a channel 52 which connects the entry port 38
with an exit port 54. The channel 52 serves to conduct the suction
force from the distal end 44 of the suction channel 97 to the exit
port 54. A single suction channel tube 56 is connected to the exit
port 54.
[0076] The suction channel tube 56 is shorter than the adhesive
channel tube 50 so that the suction channel tube 56 does not
interfere visually or functionally with the application of the
adhesive. Materials from the site of application, when suctioned,
are drawn along the exterior surface of the adhesive channel tube
50 to the orifice of the suction channel tube 56. This provides
gentle, atraumatic suction required in microsurgery.
[0077] In the apparatus of FIGS. 4 and 5, compression and expansion
of the bulb 4 is controlled by means of a leverage mechanism 58.
The leverage mechanisms 58 includes an outer lever arm 60 and an
inner lever arm 62. Both lever arm 60 and lever arm 62 turn about a
fulcrum 64.
[0078] The free end 66 of the outer lever arm 60 is positioned
adjacent the venturi opening 96 of the suction channel 16. This
placement allows the operator to switch rapidly and with a minimum
amount of movement from the control of the venturi opening 96 to
the control of the lever arm 60.
[0079] The free end 68 of the inner lever arm 62 rests against the
exterior of the flexible bulb 4. Movement of the free end 68 of the
inner lever arm 62 toward the bulb 4 will cause both compartments 6
and 8 of the bulb 4 to be compressed in equal amounts, thereby
causing a small quantity of air to be displace from each of the
compartments 6 and 8 through the liquid channels 91 and 95,
respectively. Movement of the free end 68 of the inner lever arm 62
away from the bulb 4 will allow the resilient bulb material to
return to its original dimensions.
[0080] The lateral motion of the free end 66 of the outer lever arm
60 that is transferred to the free end 68 of the inner lever arm 62
is proportional to the relative lengths of the outer lever arm 60
and inner lever arm 62. If the inner lever arm 62 is shorter than
the outer lever arm 60, as shown in FIGS. 4 and 5, then a
relatively large lateral motion of the free end 66 will be
translated into a relatively short lateral motion of the free end
68. In this manner, a relatively broad finger-controlled lateral
movement of the lever arm 60 may be used to control a relatively
small displacement of air through each of the liquid channels 91
and 95.
[0081] The pivot ends of both the outer lever arm 60 and the inner
lever arm 62 are fixed to a ratchet wheel 70. The rotational axis
of the ratchet wheel 70 is concentric with the fulcrum 64. Disposed
about a portion of the circumference of the ratchet wheel 70 is a
set of spaced ratchet teeth 72.
[0082] A pawl 74 is positioned to engage the ratchet teeth 72, and
to thereby prevent movement of the ratchet wheel 70 in a clockwise
direction as shown in FIG. 4. A finger-operable pawl release 76 is
attached to the pawl 74. The pawl release 76 provides a mechanism
for disengaging the pawl 74 from the ratchet teeth 72, thereby
allowing unrestricted movement of the ratchet wheel 70.
[0083] The combination of the ratchet wheel 70 and pawl 74 thereby
provides a mechanism for stopping movement of the lever arms 60 and
62 (as well as compression or expansion of the bulb 4) at a
plurality of fixed intervals. The size of these intervals is
dependent on the spacing between the teeth 72 and the circumference
of the ratchet wheel 70.
[0084] In the apparatus shown, the two components of the
dual-component adhesive are drawn into the channels 26 and 28 from
a filler tray 78 shown in FIG. 7. The filler tray 78 is designed to
orient the operator so that the possibility of drawing a liquid
component into the incorrect channel is minimized.
[0085] The filler tray 78 is generally of rectangular configuration
and divided by a septum 80 along the center of its longer axis into
a first container 82 and second container 84. The floor of the
first container 82 has a trough 86 which is perpendicular to the
septum 80. The floor of the second container 84 has a similar
trough 88 which is also perpendicular to the septum 80. Both
troughs 86 and 88 are located closer to one end of the filler tray
78 than the other. The resulting asymmetry of the filler tray 78
provides a useful visual orientation for the operator. Both troughs
86 and 88 slant downward toward the septum 80, so that even a small
amount of liquid placed in either container 82 or 84 will pool
towards the septum 80.
[0086] The height and width of the septum 80 are selected so that
the distal end 40 of the channel 26 will rest in the lowest part of
the trough 88 at the same time that the distal end 42 of the
channel 28 will rest in the lowest end of the trough 86. In this
way, even small amounts of liquid in the containers 82 and 84 will
be directed to the distal ends 40 and 42 of the channels 26 and 28.
The height of the septum 80 is selected so that the distal end 44
of the suction channel 97 rests above the septum 80 when fluid is
drawn from the filler tray 78. The points at which the distal ends
40 and 42 of the channels 26 and 28 rest adjacent to the septum 80
are the lowest of all points in the filler tray 78. In this way any
fluid placed in either container 82 or 84 of the tray 78 will pool
towards the distal ends 40 and 42 during the filling of channels 26
and 28.
[0087] The filler tray 78 is anchored to a heavyweight base 90 to
help prevent the liquid contents of the filler tray from being
spilled.
[0088] To operate the device, the outer lever arm 60 is first
pressed toward the housing 2 so that the fulcrum 64 rotates in a
counterclockwise direction as shown in FIG. 4. The free end 68 of
the inner lever arm 62 is thereby forced against the exterior of
the bulb 4, compressing the bulb. The free end 68 of the lever arm
62 will be held in place against the bulb 4 by means of the pawl 74
engaging the ratchet teeth 72 of the ratchet wheel 70.
[0089] The distal ends 40 and 42 of the fluid channels 26 and 28
are then placed into the corresponding containers 82 and 84 of the
filler tray 78. The pawl release 76 is operated to disengage the
pawl 74 from the ratchet teeth 72. The ratchet wheel 70 is thereby
allowed to rotate freely about the fulcrum 64.
[0090] In the absence of pressure exerted by the free end 68 of the
inner lever arm 62, the resilient flexible material of the bulb 4
will return to its initial configuration, creating a pressure
differential between the interior of the bulb 4 and the ambient
atmosphere. Atmospheric pressure will force the liquid contents of
the filler tray containers 82 and 84 into the corresponding fluid
channels 40 and 42 in a volume approximately equal to that of the
air originally displaced from the bulb 4.
[0091] The distal ends 40 and 42 of the fluid channels 26 and 28
are then removed from the containers 82 and 84 of the filler tray
78. The mixing tip 94 is then attached to the fluid channels 26 and
28 and the suction channel 97. The mixing tip 94 is brought to the
work surface. If cleaning of the surface is desired immediately
before application of the adhesive, then the venturi opening 96 of
the suction channel 16 is closed off by the operator's finger. The
suction force created by a remote vacuum attached to the proximal
end of the suction channel is transmitted via suction channel 16 to
the work surface. Fluid and debris on the work surface is thereby
removed through the suction channel 16.
[0092] The precise amount of adhesive deposited on the work surface
is accurately controlled via the leverage mechanism 58. As the
outer lever arm 60 is pressed, the ratchet teeth 72 will rotate
past the pawl 74. As each ratchet tooth passes the pawl 74, an
audible "click" will be heard.
[0093] The ratchet teeth 72 may be positioned so that each "click"
represents the deposit of a predefined amount of adhesive. For
example, if the ratchet teeth 72 are positioned so that each
"click" corresponds to the deposit of one microliter of adhesive,
then an operator wishing to apply precisely three microliters of
adhesive will press the outer lever arm 60 until three "clicks" are
heard. The operator's visual attention to the work area thereby
remains uninterrupted.
[0094] At any point during the procedure, application of the
adhesive may be halted by merely removing finger pressure from the
outer lever arm 60. The lever arm 60 will be maintained in position
by the pawl 74 engaging the ratchet teeth 72.
[0095] In any event, the fibrinogen adhesive of the invention is
applied along with a calcium/thrombin composition as understood by
the art by any suitable application means.
* * * * *