Centrifuge with annular filter

A method and a device for separating a component, such as fibrin I from blood, by centrifugation. The method involves feeding of blood admixed an anticlotter to a first annular chamber in a device, where the annular chamber is defined by a cylindrical outer wall and a cylindrical inner wall, both walls extending coaxially about a common axis, as well as by a top wall and a bottom wall, where the top wall or the bottom wall is formed by a piston body displaceable within the first chamber. The method involves furthermore a centrifugation of the device about the said common axis followed by a resulting liquid fraction being transferred while influenced by the piston body to a second chamber defined by an outer cylindrical wall, which extends coaxially with said common axis. As a result the liquid fraction present in the second chamber is caused to be further separated by a continued centrifugation and addition of suitable compositions promoting the separation and returning of the non-utilizable portion to the first chamber. In addition, the portion of the liquid fraction remaining in the second chamber is transferred to a liquid-receiving container through a filter optionally after addition of a solvent. The portion of the liquid fraction remaining in the second chamber prior to the transfer to the liquid-receiving container is transferred to a third chamber coaxially accommodated with the other chambers, and the liquid now present in said third chamber is caused to pass through an annular filter during the centrifugation so as to enter an annular outer compartment which is adapted to be connected to the liquid-receiving member.

TECHNICAL FIELD 
The invention relates to a centrifuge device for separating a component, 
such as fibrin monomer from plasma, said method involving treating of 
plasma with one or more reagents wherein said reagents are delivered to a 
suitable reaction chamber containing said plasma and where such reagents 
are therefore removed from a desired product by way of novel centrifugal 
filtration device and method. 
BACKGROUND ART 
EP-PS No. 592,242 describes methods and compositions for a completely novel 
fibrin sealant involving contacting a desired site with a composition 
comprising fibrin monomer and converting this monomer to a fibrin polymer 
concurrently with the contacting step. The term "fibrin" is defined as 
fibrin I, fibrin II, and/or des .beta..beta. fibrin. 
Further a method is known from U.S. Pat. No. 5,603,845 for separating a 
component, such as fibrin monomer from blood. This method for separating 
the components of a liquid containing several components of a varying 
specific gravities involves the steps of the blood being collected in a 
first chamber of a device, said chamber being defined by a substantially 
axially symmetrical outer and inner wall. The blood is subjected to a 
centrifugation by way of rotation of the device about the axis of symmetry 
of the chamber so as to establish a concentric interface between the 
components of the blood. At least one of the components of the blood such 
as plasma is subsequently transferred to a second chamber in the device 
preferably by way of reduction of the volume of the first chamber during a 
continued centrifugation of the device. The substantially axially 
symmetrical inner wall is provided in the first chamber so as to ensure 
that all the blood is subjected to a centrifugal rotation necessary for 
the separation. This inner wall is of a radius adapted to the desired 
speed of rotation. 
In the second chamber a fraction with non-crosslinked fibrin polymer is 
separated from the plasma by means of a suitable enzyme and subsequently 
redissolved into fibrin monomer and transferred to a syringe through a 
filter by reducing the volume of the second chamber. 
It turned out, however, that the separation of a component, such as fibrin 
I from blood, only by way of filtration in a device of the above type does 
not provide a satisfying result. This is mainly due to the fact that it is 
difficult to ensure a satisfying separation of the fraction containing 
fibrin I in the second chamber, and accordingly a relatively high amount 
of the content in the blood of fibrin I is lost during the following 
transfer of a fluid fraction from the second chamber to the first chamber 
during the succeeding step of the method. 
Also, in the earlier fibrin monomer method, the above-described treatment 
of fibrinogen within the plasma with a suitable enzyme produced the 
non-crosslinked fibrin polymer in the form of a thick gel mass at the 
bottom of the second chamber. To provide the desired fibrin monomer 
solution, a significant amount of redissolving buffer combined with 
substantial agitation was required. This resulted in several drawbacks. 
First, preferred fibrin monomer methods, e.g., for use as a fibrin sealant 
as in EP 592,242, require concentrated fibrin monomer solutions, and the 
large amount of redissolving buffer or solvent required to dissolve the 
gel mass provided dilute solutions which do not work as well. Further, the 
substantial agitation required to dissolve the gel mass into a fibrin 
monomer solution can cause damage to the device and to the fibrin itself. 
A co-pending application entitled "Method and Device for Separating a 
Component Such as Fibrin I From Blood Plasma", U.S. Ser. No. 08/566,348 
now allowd, discloses an invention including a method which provides for 
the separation of non-crosslinked fibrin polymer from a plasma fraction in 
a cylindrical chamber carried out during centrifugation whereby the 
non-crosslinked fibrin polymer is deposited on the outer wall of the 
chamber, whereafter the remaining fluid fraction collected in the chamber 
is removed from the chamber, and that the fraction with non-crosslinked 
fibrin polymer remaining in the chamber substantially deposited on the 
wall is caused to be dissolved by addition of a solvent and by centrifugal 
agitation. 
Since the treatment of the plasma with the enzyme is carried out during 
continued centrifugation, the centrifugal force upon the resulting 
non-crosslinked fibrin polymer provides that it is precipitated as a thin 
gel film which substantially sticks to the circumferential walls of the 
chamber. The remaining plasma liquid deposits at the bottom of the chamber 
when the centrifugation is stopped and can be removed by any convenient 
means. The desired fibrin monomer solution is thereafter provided by 
introducing a suitable redissolving buffer solution into the chamber and 
subjecting the buffer in the gel-coated chamber to centrifugal agitation. 
This method provides advantages over prior methods. First, the 
redissolving of the non-crosslinked gel by the buffer solution is 
extremely efficient due in part to the large surface area of the same 
volume of fibrin gel compared to the fibrin gel mass provided in prior 
methods. Accordingly, the gel can be dissolved with small amounts of 
redissolving buffer resulting in a desirably concentrated fibrin monomer 
solution. Further, the action of the centrifugal agitation on the buffer 
solution within the gel-coated chamber is a comparatively gentle method 
causing no damage to the equipment or to the fibrin monomer product. 
The copending invention also includes a method involving feeding of blood 
preferably in the presence of an anticoagulant to a first annular chamber 
in a device, where the annular chamber is defined by a cylindrical outer 
wall and a cylindrical inner wall, both walls extending coaxially about a 
common axis, as well as by a top wall and a bottom wall, where the top 
wall or the bottom wall is formed by a piston body displaceable within the 
first chamber, said method further involving a centrifugation of the 
device about the said common axis to substantially separate blood into a 
cell fraction and a plasma fraction followed by the resulting plasma 
fraction being transferred while influenced by the piston body to a second 
chamber defined by an outer cylindrical wall, which extends coaxially with 
said common axis, whereby a fraction with non-crosslinked fibrin polymer 
is caused to be separated in the second chamber while a suitable enzyme is 
being added. This method is characterized in that the 
fibrinogen-containing plasma fraction is subjected to the enzyme during 
centrifugation so that the resulting non-crosslinked fibrin polymer is 
deposited on the cylindrical outer wall of said second chamber, whereafter 
the fluid fraction collected at the bottom of the second chamber is 
transferred while influenced by the piston body to the first chamber, and 
that the fraction with non-crosslinked fibrin polymer remaining in the 
second chamber substantially deposited on the cylindrical wall is caused 
to be dissolved by addition of a solvent and by centrifugal agitation. 
Thereafter the enzyme can be removed, if desired, and the so-produced 
fibrin monomer solution is transferred to any desired receiving container. 
Accordingly, an aseptic condition for collecting the solution is easily 
maintained. After the fibrin monomer has been redissolved, it can be 
transferred to a receiving container, such as a syringe for further use as 
described in the prior art. Before the transfer, the enzyme can be removed 
by any convenient means. 
The above copending application further discloses a device for separating 
components from a liquid by way of centrifugation about a central axis of 
rotation comprises a first annular chamber defined by an outer cylindrical 
wall and an inner cylindrical wall, both walls being concentrically 
accommodated about said axis of rotation, as well as by a top wall and a 
bottom wall, where the bottom wall is formed by a piston body displaceable 
within said first chamber, said device further comprising a second chamber 
communicating with the first chamber through a first conduit and being 
defined by an outer cylindrical wall concentrically accommodated about the 
axis of rotation and by said piston body and a bottom wall, where said 
second chamber is adapted to be positioned below the first chamber during 
the centrifugation, and where said device also comprises blood feeding 
means for feeding blood to the first chamber and composition feeding means 
for feeding composition promoting the separation as well as receiving 
means for the connection of at least one liquid-receiving container, where 
the receiving means communicate with the second chamber through a second 
conduit. In a preferred embodiment, the piston rod comprises the inner 
wall of the first chamber. 
This inventive device for carrying out the method according to the 
copending invention is characterized in that the first conduit comprises 
at least one channel extending between an opening at the top wall of the 
first chamber and an opening at the bottom wall of the second chamber. 
As a result a device is provided which is relatively simple and which 
independent of the position of the piston ensures an easy and fast 
transfer of the fractions in question from one chamber to the other 
chamber, and especially of the fluid fraction from the second chamber to 
the first chamber after the separation of the fibrin-l-containing 
fraction. The latter is especially due to the fact that the fluid is 
automatically concentrated at the bottom of the second chamber when the 
centrifugation is stopped, whereby it can be easily transferred to the 
first chamber by the piston being moved. 
According to the copending invention it is particularly preferred that said 
at least one channel extends through the interior of the outer cylindrical 
wall in both the first and the second chamber with the result that the 
device is particularly simple and easy to manufacture. 
Further, the opening of the channel at the bottom wall of the second 
chamber may be centrally accommodated in the chamber in connection with a 
recess formed by the bottom wall. As a result, the fluid fraction in 
question is easily and quickly guided directly to the inlet opening of the 
channel. Alternatively, each channel may be formed by a pipe extending 
rectilinearly through the piston body and being secured at the ends in the 
top wall of the first chamber and the bottom wall, respectively, of the 
second chamber where it communicates with channel portions ending in the 
respective chamber. 
Additionally, the first and the second chamber may in a particularly simple 
manner comprise a common outer cylindrical wall shaped by an outer and an 
inner cylinder sealingly fitting within one another and defining 
therebetween an axially extending channel, and the cylinders may be 
terminated at one end by an end wall comprising an opening allowing 
passage of a piston rod connected to the piston body, said piston body 
forming the bottom wall of the first chamber and separating said first 
chamber from the second chamber, and where the channel extends between the 
end walls of the cylinders to an opening immediately adjacent the piston 
rod. 
In using such a device and method, suitable reagents for facilitating the 
separation and treatment of desired components within the blood plasma 
were preloaded into the second chamber. For example, EP 592,242 describes 
that the biotin-avidin capture system can be conveniently used to remove 
the batroxobin from the desired solution. It is required that the biotin 
batroxobin be present in the second chamber to react with the fibrinogen 
within the plasma and convert it to a fibrin monomer (which immediately 
converts to a fibrin polymer). In order to thereafter capture the 
biotinylated batroxobin using the biotin-avidin system, avidin which is 
bound, for example, to agarose must also be present in the second chamber. 
In a closed, automated centrifuge device, these agents need to be loaded 
into the device prior to blood processing. Preloading of the biotinylated 
batroxobin and avidin agarose into the same chamber has provided 
difficulties since the high affinity of the avidin for the biotin, which 
is relied upon for enzyme capture, prevents sufficient quantities of the 
enzyme from first reacting with the fibrinogen as is required. 
A second copending application filed concurrently herewith entitled 
"Centrifuge Reagent Delivering System", U.S. Ser. No. 08/566,195, allowed, 
describes a device which renders it possible to easily place one or more 
reagents inside a reaction chamber and to release such reagents in a 
desired sequence. Preferably when used in a device of the type described 
in the aforementioned copending application, reagents such as an enzyme 
and an enzyme-capture composition can be released as desired. In 
satisfaction of the foregoing object there is according to the invention 
provided a device which is characterized in that a capsule is accommodated 
in the second chamber and comprises a plurality of compartments for 
receiving respective compositions promoting the separation, and that the 
capsule comprises closing means closing said compartments and while 
influenced by the piston being adapted in sequence to open for the release 
of the contents of the compartments. 
Such a capsule renders it possible in a simple and easy manner to feed the 
substances necessary for the separation of fibrin I, said capsule 
preferably being provided with these substances in advance. In addition, 
the provided compartments allow a uniform predetermined apportion of the 
amount in question. The batroxobin is preferably placed in one compartment 
in chemical relationship with biotin providing that the enzyme batroxobin 
can be easily captured after the use by means of avidin, which is 
therefore placed in the second compartment in chemical relationship with 
agarose in form of relatively large particles. The high affinity of the 
biotin for the avidin provides that complexed biotinylated 
batroxobin/avidin agarose particles are subsequently readily removed by 
filtration from the fibrin I solution. The placing of the two substances 
in their respective compartment renders it also possible to easily dose 
the substances at the desired times by influencing the piston. The above 
substances or compositions, biotin-batroxobin, respectively, and 
avidin-agarose can be used in any convenient form, e.g., lyophilized 
powder form. 
According to the copending reagent delivery system invention it is 
particularly preferred that the capsule comprises a central hub coaxially 
mounted in the interior of the second chamber and carrying three mutually 
spaced radial disks forming partitions in the compartments and being of a 
substantially identical outer circumferential contour, and that the 
closing means are formed by a sleeve-shaped body displaceably, but 
sealingly surrounding the radial disks. 
For activating the sleeve-shaped displaceable body the piston may according 
to the invention advantageously comprise a downward skirt cooperating with 
the sleeve-shaped body on the capsule so as to displace said sleeve-shaped 
body stepwise whereby said body in sequence opens for release of the 
contents of said compartments inside the capsule. 
According to the copending reagent delivery system invention the capsule 
may be accommodated in connection with an axial passage to an adjacent 
third chamber, the outer side of the sleeve-shaped body of the capsule 
sealingly abutting the side wall of the axial passage at least after an 
initial displacement of the body, whereby the lowermost partition of the 
capsule allows a free passage of liquid from the second chamber to the 
third chamber after a final displacement of the sleeve-shaped body caused 
by the piston out of its engagement with the circumference of the 
lowermost partition. In this manner the capsule forms furthermore part in 
an advantageous manner of the device and assist said device in its further 
operation during the separation of the fibrin I. 
So as further to form an integrated part of the device, the hub of the 
capsule may according to the invention comprise an axial, through passage 
and be secured on an upward projection centrally positioned in the bottom 
of the lower, third chamber, said through passage at the bottom liquidly 
communicating with the outer annual compartment of the third chamber 
through a channel system, and the upper end of the hub may be adapted to 
be sealingly connected to an axial passage in the piston body so as to be 
connected to a liquid-receiving container securable thereto. 
To remove the one or more reagents from the desired product solution the 
resulting fibrin-l-containing fluid fraction is chamber into a syringe 
through a filter while influenced by the piston. As a result, the solution 
of fibrin I is forced through the filter while the enzyme and other 
substances admixed for accelerating the separation are retained by the 
filter. The resulting yield of fibrin I is, however, not completely 
satisfying when compared to the amount of fibrin I present in a blood 
sample. 
SUMMARY OF THE INVENTION 
An object of the invention is therefore to provide a method rendering it 
possible to achieve a higher yield of fibrin I by means of a device of the 
type dealt with. 
In satisfaction of the foregoing object there is according to the invention 
provided a method which is characterized in that the portion of the liquid 
fraction remaining in the second chamber prior to the transfer to the 
liquid-receiving container is transferred into a third chamber coaxially 
accommodated with the other chambers, and that the liquid now present in 
said third chamber is caused to pass through an annular filter during the 
centrifugation so as to enter an annular outer compartment which is 
adapted to be connected to the liquid-receiving member. As a result, the 
fibrin I-solution can be passed through a filter under the influence of 
the centrifugal force which is considerably more effective than filtration 
by means of the piston. 
The invention relates furthermore to a device for carrying out the above 
method. The inventive device comprises a first annular chamber defined by 
an outer cylindrical wall and an inner cylindrical wall, both walls being 
concentrically accommodated about the axis of rotation, and by a top wall 
and a bottom wall, where the top wall or the bottom wall is formed by a 
piston body displaceable within the first chamber, said device further 
comprising a second chamber communicating with said first chamber through 
a first conduit and being defined by an outer cylindrical wall 
concentrically accommodated about the axis of rotation, said bottom wall 
of the first chamber and another bottom wall, where the second chamber is 
adapted to be placed below the first chamber during the centrifugation, 
and where said device also comprises blood feeding means for feeding blood 
to the first chamber and composition feeding means for feeding 
compositions promoting the separation as well as receiving means for the 
connection of at least one liquid-receiving container, said receiving 
means communicating with the second chamber through a second conduit. 
This device is according to the invention characterized in that the second 
conduit communicates with the second chamber through a third chamber 
coaxially accommodated relative thereto and comprising a passage to the 
second chamber which can be opened from the outside, that the third 
chamber comprises an inner compartment and an outer annular compartment, 
said compartments being interconnected through a radially extending 
circumferential passage, in which an annular filter is arranged for 
preventing passage of liquid containing undesired ingredients used for 
promoting the separation. 
According to the invention the passage between the second and the third 
chamber may be coaxially arranged relative to the two chambers and is 
closed by means of a capsule which is disclosed in the aforementioned 
copending patent application entitled "Centrifuge Reagent Delivery 
System". This capsule comprises a central hub coaxially mounted in the 
second chamber and which carries a plurality of spaced apart radial disks 
forming partitions in a plurality of compartments in the capsule, where 
the disks are of an identical outer circumferential contour, that 
outwardly the compartments are closed by means of a sealing, displaceably 
mounted, sleeve-shaped body, the outer side of which is adapted to 
sealingly abut the side wall of the axial passage, the lowermost 
partition-forming disk of the capsule allowing a free passage of liquid 
from the second chamber into the third chamber by an axial displacement of 
the sleeve-shaped body out of its engagement with the circumference of the 
lowermost partition while influenced by the piston. As a result, an easy 
and simple access is obtained to the filter in question by means of a 
capsule which is already used for feeding the substances necessary for 
promoting the separation of fibrin I inside the second chamber. 
This capsule is preferably activated by the piston body comprising a 
downward skirt extending coaxially with the sleeve-shaped body of the 
capsule and being adapted to engage said body when the piston body is 
pressed down so as thereby in sequence to open at suitable moments for the 
respective compartments in the capsule and finally to open for the passage 
of liquid from the second chamber into the third chamber. 
In order to facilitate the transferring of the fibrin I-solution to a 
liquid-receiving container, such as a syringe, the hub of the capsule may 
according to the invention comprise an axial, through passage and be 
secured on an upward projection centrally arranged in the bottom of the 
lower third chamber, said through passage liquidly communicating with the 
outer annular compartment of the third chamber through a channel system, 
and the upper end of the hub may be adapted to be sealingly connected to a 
passage in the piston body so as to be connected with a liquid-receiving 
container securable therein. 
Finally according to the invention the hub of the capsule may be secured to 
the projection in the bottom of the third chamber by means of a sleeve, 
which at each end surrounds the hub and the projection, respectively, and 
the sleeve may comprise a circumferential, outwardly projecting wall 
portion, whereby the said annular filter is secured between the outer 
circumference of said wall surface and the bottom wall of the second 
chamber, whereby the outwardly projecting wall portion of the sleeve is 
accommodated at a distance from the bottom of the third chamber and 
thereby forms the connection between the outer annular compartment and an 
axially extending channel in connection with the through passage of the 
hub and extending between the outer side of the projection and the 
adjacent inner side of the sleeve. The resulting device is particularly 
simple. 
The methods of the present invention deal with improved processes for 
separating and isolating an individual blood component or a solution 
containing such a component. However, the present method is suitable for 
any procedure adaptable to a cylindrical centrifuge, wherein a first 
solution is treated with one or more catalysts or reagents during 
centrifugation. Other blood procedures which could benefit from such a 
method include, but are not limited to the isolation of any blood 
component, such as 
platelet-rich plasma, 
platelet concentrate, 
cryoprecipitated fibrinogen, 
other proteins within plasma such as thrombin, fibronectin and the like. 
Preferably the blood is from a single donor and most preferably the blood 
is from the same person to whom the blood component will be administered . 
While the present methods are hereinafter described in terms of producing a 
fibrin monomer solution, the scope of the invention as will be appreciated 
by those skilled in the art, should not be so limited. 
As used herein, the term "centrifugal agitation" refers to the motion of 
the device where the redissolving buffer solution is introduced to 
redissolve the intermediate product, such as non-crosslinked fibrin 
polymer gel, from the outer chamber walls. Such motion or centrifugal 
agitation may include centrifugation to ensure that all of the exposed 
surface area of the gel is subjected to the redissolving solution, and 
includes preferably such a centrifugation followed by stop-and-start 
rotations in the same direction and/or stop-and-start rotations in 
opposite directions. Typical centrifugal agitations include, but are not 
limited to, 5-30 second spins, preferably 5-10 second spins, at 
2,000-5,000 RPM in repeated forward/reverse cycles for any desired length 
of time. In the present methods, 5-10 second spins at about 3,000 RPM in 
repeated forward/reverse cycles for 1-2 minutes is preferred. As mentioned 
above, this can be preceded by a somewhat longer spin, e.g., 20 seconds or 
more to initially distribute the solvent. 
The term "fibrin" as used herein refers to fibrin I, fibrin II or des 
.beta..beta. fibrin. 
The present device incorporating the centrifugal annular filter system 
disclosed herein provides an efficient and accurate method for recovering 
one or more reagents from a desired product solution. This is especially 
critical in closed, self-contained, automated centrifuges for use in blood 
separation techniques wherein two or more reagents are required to be 
introduced into a reaction chamber in a sequential manner and therefore 
removed. In the preferred methods and devices described herein for 
providing a fibrin monomer-containing solution, e.g., for use in a novel 
fibrin sealant, the sequential introduction of biotinylated batroxobin 
followed by avidin agarose into the plasma containing chamber provides a 
highly sophisticated method of preparing such a solution.

The present device is a single, closed automatable device capable of 
converting whole blood into desired blood components preferably autologous 
components useful, for example, as fibrin sealants. 
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION 
Preferably the present centrifuge annular filter system is employed with a 
device as covered in the above referenced copending applications and it is 
therefore described below with response to such a device. However, it 
should be understood that it could be employed in any reaction chamber 
device requiring removal of one or more reagents. 
The device of FIG. 1 according to the invention is built of parts 
substantially presenting rotation symmetry and implying that the device 
can be placed in a centrifugation apparatus in an easy manner known per se 
so as to be centrifuged about a central axis 1. In this FIG. 1, a 
preferred embodiment of the device comprises an outer container 2 and an 
inner container 3 being such that they completely fit into each other and 
everywhere closely abut one another apart from the portion where an 
axially extending intermediary channel 4 is provided. The channel 4 is 
provided by a groove shaped in the inner container 3. The two containers 2 
and 3 comprise their respective top portions 5 and 6, respectively, which 
define a central opening 7 allowing passage of a piston rod 8. About the 
opening 7, the two containers comprise axially extending parts 9 and 10, 
respectively, which extend close to the hollow piston rod 8 in a direction 
away from the interior of the containers. The outer container 2 abuts the 
hollow piston rod along a short radially extending flange 11 provided with 
a recess 12 receiving a sealing ring 13. 
As illustrated in FIG. 1, the channel 4 continues between the inner and the 
outer container all the way from the outer cylindrical walls of the inner 
and the outer container along the top portions 5, 6 and the axial parts 9 
and 10 to the opening immediately below the sealing ring 13 in the opening 
7. The axial part 10 of the inner container 3 abutting the opening 7 is 
dimensioned such that a narrow, but free passage exists to the interior of 
the containers 2 and 3 about the hollow piston rod 8. 
The outer container 2 comprises a cylindrical part of a uniform diameter, 
cf. FIG. 1. Downwardly when seen relative to the drawing, this part 
continues into a cylindrical part 14 of a slightly larger diameter through 
a short transition part 15 forming a frusto-conical inner surface 16. The 
inner container 3 ends at the location where the transition part 15 of the 
outer container 2 continues into the cylindrical part 14 of a larger 
diameter. The lower end of the inner container 3 comprises an outer 
surface 17 of a frusto-conical form matching the form of the 
frusto-conical surface 16 on the inner side of the outer container 2. A 
outer and an inner annular disk 19 and 20, respectively, are provided 
immediately below the lower end of the inner container 3, which ends in a 
radial surface 18. These disks closely abut one another apart from the 
fact that they define therebetween a channel 21 extending in an axial 
plane from a central opening 22 and forwards to the inner side of the 
outer container 2, where the channel 21 communicates with the channel 4 
between the outer container 2 and the inner container 3 through an axially 
extending part 23. The channel 21 and the axial channel part 23 are 
suitably provided by means of a groove in the side of the inner disk 20 
facing the outer disk 19. The two disks 19 and 20 are shaped with such an 
oblique course that they comprise substantially inner and outer 
frusto-conical surfaces, cf. FIG. 1, and thereby incline downwards towards 
the central opening 22. FIG. 1 also shows that the inner disk 20 comprises 
a radial surface 24 abutting the adjacent radial surface 18 on the inner 
container 3. The radial surface 24 of the inner disk 20 is provided with a 
recess 25 for receiving a sealing ring 26. 
The two disks 19 and 20 are maintained in position in abutment against the 
radial surface 18 of the inner container 3 by means of a cover 27 closing 
the outer container in the downward direction. This cover 27 comprises a 
circumferential sleeve-shaped part 28 adapted to closely abut the inner 
side of the outer container 2, to which it is secured in a suitable 
manner, such as by way of a snap-action by engagement between a 
circumferential rib 29 on the outer side of the sleeve 28 and a 
corresponding circumferential groove 30 on the inner side of the outer 
container 2. A sealing connection is ensured by means of a sealing ring 31 
in a circumferential recess 32 at the outer periphery of the outer disk 
19. The cover 27 comprises furthermore a relatively thin wall 32 adapted 
to form the lower bottom of the device in the position shown in FIG. 1. 
This wall 32 extends substantially along a course parallel to the outer 
and the inner disk 19 and 20 in such a manner that the wall 32 extends 
from the inner side of the sleeve 28 in a portion adjacent the disks 19 
and 20 and downwards towards a portion substantially on a level with the 
lower rim 33 of the outer container 2. In order to reinforce this 
relatively thin wall 32, a reinforcing radial rib 34 is provided at 
regular intervals, only one of said ribs appearing from FIG. 1. This rib 
34 is shaped partly with a portion placed outside the wall 32 and partly 
with a portion placed inside the wall 32, cf. FIG. 1. The latter portion 
is designated the reference numeral 35 and is shaped such that it abuts 
the bottom side of the outer disk 19 with the result that it assists in 
maintaining the disks 19 and 20 in a reliable position. 
A partition means 36 is squeezed between the outer disk 19 and the cover 
27. This partition means 36 comprises a central pipe length 37. This pipe 
length is mounted on a pin 38 projecting axially inwards and being shaped 
integral with the wall 32 of the cover 27. This pipe length 37 is shaped 
integral with a circumferential wall disk 39 extending outwardly from the 
pipe length 37 in such a manner that initially it inclines slightly 
downwards towards the wall 32 of the cover 27 whereafter it extends along 
a short axial course so as to continue into a course extending 
substantially parallel to the wall 32 of the cover. The wall disk 39 ends 
in a short radially extending periphery 40 resting on a shoulder 41 on the 
rib portions 35 on the cover 27. An annular filter unit 42 is squeezed 
between the outer periphery 40 of the wall disk 39 and the bottom side of 
the outer disk 19. This annular filter unit 42 abuts a substantially 
radially shaped surface 43 on the adjacent outer side of the outer disk 
19. 
In order to ensure a stability in the partition means 36, reinforcing 
radial ribs designated the reference numeral 44 are furthermore 
accommodated between the pipe length 37 and the wall disk 39. 
The reagent delivery system of the present invention comprises a capsule 
designated the general reference numeral 45 is secured in the end opposite 
the cover 27 of the pipe length 37 of the partition means 36. Such a 
capsule is suitable for selectively releasing agents into the second 
chamber 75. This capsule comprises an elongated pipe length 46 shaped 
integral with a radial ring 47 and carrying two additional radial rings 48 
and 49. These radial rings 48 and 49 are secured by way of interference 
fit on their respective side of the fixed ring 47. The loose rings 48 and 
49 are accommodated at their respective distance from the fixed ring 47 by 
means of circumferential shoulders 50 and 51, respectively, on the pipe 
length 46. The three disks 47, 48, and 49 are all of the same outer 
diameter and carry along their respective peripheries a circumferential, 
displaceably mounted sleeve 52. 
As illustrated in the drawing, the lower disk 49 abuts the upper end of the 
pipe length 37 of the partition means 36, whereby the position of the 
capsule 45 in the axial direction is determined. This position is 
furthermore determined in such a manner that when displaced in the axial 
direction the displaceable sleeve 52 of the capsule enters a sealing 
engagement by its lower end, cf. the drawing, with the innermost edge 53 
on the outer disk 19 in the central opening 22. In this position of the 
sleeve 52, a communication still exists between the space inside the inner 
disk 20 surrounding the sleeve 52 and the inlet opening to the channel 21 
between the outer disk 19 and the inner disk 20. The axial length of the 
displaceable sleeve 52 is adapted such that the engagement with the outer 
disk 20 occurs before the upper end, cf. the drawing, of the sleeve 52 
disengages the fixed ring 47 during the axial downward displacement of 
said sleeve 52. The inner diameter of the sleeve 52 is also adapted to the 
outer diameter of the axially extending part of the wall disk 39 of the 
partition means 36 in such a manner that a continued downward displacement 
of the sleeve 52 towards the cover 27 causes said sleeve 52 to fixedly 
engage the partition means 36 once it has disengaged the outer disk 19. 
The length of the axial part of the partition means 36 corresponds also to 
the axial length of the sleeve 52 in such a manner that said sleeve 52 in 
the lowermost position is substantially completely received by the 
partition means 36. 
As illustrated in the drawing, the hollow piston rod 8 comprises a 
circumferential piston 55 inside the outer container 2 and the inner 
container 3, said piston 55 sealingly engaging the inner side of the inner 
container 3 through a sealing ring 56. 
A Luer-coupling 57 is shaped inside the hollow piston rod for receiving a 
conventional syringe 58 with a piston-acting plug 59 for acting on the 
content of the syringe 58. The coupling 57 is shaped substantially as a 
length of pipe communicating with a central opening 61 in the piston 55 
through a frusto-conical portion 60. The length of pipe 57 is provided 
with a radially inwardly projecting web 62 for directing the fluid leaving 
the syringe 58 away from an axial path and thereby round the length of 
pipe 46 therebelow inside the capsule 45. The latter length of pipe 46 is 
of such a length and such dimensions that it can sealingly engage the 
length of pipe 57 inside the hollow piston rod 8 when the piston 55 is in 
its lowermost position near the cover 27. In order to promote the above 
sealing connecting, the inner side of the length of pipe 57 is formed with 
a gradually decreasing diameter at the end adjacent the piston 55. 
An axially projecting skirt 63 is formed integral with the piston 55 about 
the central opening 61 of said piston. This skirt 63 is shaped with such a 
diameter and such a length that by a suitable displacement of the piston 
55 it can activate the above displacement of the displaceable sleeve 52 of 
the capsule 45 into the said positions in which it engages the inner rim 
53 of the central opening 22 through the two disks 19 and 20 followed by 
an engagement of the partition means 36. 
A resilient, annular lip sealing means 64 is as indicated secured about the 
hollow piston at the top inside the containers 2 and 3, cf. FIG. 1. This 
lip sealing means 64 is adapted to prevent an undesired passage of fluid 
from the interior of the containers 2 and 3 to the channel 4, but it 
allows passage of fluid when a force is applied through the piston 55. 
As indicated at the top of FIG. 1, a connection is provided to a hose 65 
through an opening 66 in the outer and the inner container 2 and 3, 
respectively. This connection is known and therefore not shown in greater 
detail, but it allows an interruption of the connection to the hose when 
desired. In addition, an air-escape opening with a suitable filter is 
provided in a conventional manner and therefore neither shown nor 
described in greater detail. 
A passage 69 is provided from the area between the partition means 36 and 
the cover 27 and all the way upwards through the interior of the length of 
pipe 37 of the partition means 36 and through the interior of the length 
of pipe 46 of the capsule 45. This passage 69 allows a transfer of fluid 
to the syringe 58 from said area when the latter length of pipe 46 is 
coupled to the length of pipe 57 in the interior of the piston rod 8. The 
passage 66 is provided at the lowermost portion of the pin 38 in the cover 
27 by said pin 38 being shaped with a plane, axial surface, said pin being 
of a substantially circular cross section. As a result, a space is 
provided between the pin and the adjacent portion of the inner side of the 
length of pipe 37. An area 67 is provided immediately above the pin 38 
where the partition means 36 presents a slightly reduced inner diameter. 
In this manner it is possible to place a small filter 68 immediately above 
the said area, cf. FIG. 1, whereby the fluid must pass said filter before 
it enters the length of pipe 46 of the capsule 45. 
The described device comprises a first annular chamber 70 defined inwardly 
by the hollow piston 8 forming a cylindrical inner wall 71, and outwardly 
by a cylindrical outer wall 27 formed by the outer container 2 and the 
inner container 3. When in the conventional use position, cf. FIG. 1, the 
annular chamber 70 is upwardly defined by a top wall 73 formed by the 
bottom 5 and 6, respectively, of the outer container 2 and the inner 
container 3. Downwardly, the annular chamber 70 is defined by a bottom 
wall 74 formed by the piston 55. A second chamber 75 is defined below the 
piston 55, said second chamber outwardly being defined by the same 
cylindrical outer wall 72 as the first chamber 70. Downwardly, the second 
chamber 75 is defined by a second bottom wall 76 formed by the outer disk 
19 and the inner disk 20. The capsule 45 is centrally accommodated in the 
interior of the second chamber 75. A third chamber 77 is provided below 
the said second bottom wall 76, and this third chamber 77 is defined by 
the partition means 36 and the annular filter unit 42. In addition, this 
third chamber 77 communicates with the second chamber 75 through the 
passage formed by the central opening 22 in the outer disk 19 and the 
inner disk 20. Finally, a fourth chamber 78 is provided below the 
partition means 36, said fourth chamber 78 being defined downwardly by the 
wall 32 of the cover 27 and furthermore by portions of the sleeve 28 of 
the cover 27 and the bottom side of the outer disk 19. 
As described above, the described device is primarily suited for separation 
of a component, such as fibrin monomer from blood, and for this purpose 
the second chamber 75, and preferably the upper chamber 80 of the capsule 
46, is in advance filled with a suitable enzyme, such as batroxobin. As is 
understood from EP-PS No. 592,242, any thrombin-like enzyme can be 
employed. Such enzymes include thrombin itself or any other material with 
a similar activity, such as Ancrod, Acutin, Venyyme, Asperase, Botropase, 
Crotabase, Flavorxobin, Gabonase, and the preferred Batroxobin. Batroxobin 
can be chemically bound to biotin, which is a synthetic substance allowing 
the batroxobin to be captured in a conventionally known manner by means of 
avidin in an avidin-agarose composition. Accordingly, avidin-agarose is 
found in the lowermost chamber 81 of the capsule. Both the 
biotin-batroxobin composition and the avidin-agarose composition are 
relatively easy to fill into the respective chambers 80 and 81 inside the 
capsule 45 before said capsule is placed inside the device. 
Finally, a syringe 58 is arranged, said syringe containing a pH-4 buffer 
prepared from an acetate diluted with acetic acid and suited for receiving 
fibrin I. 
Another buffer known from the prior art can also be used. The redissolving 
buffer agent can be any acid buffer solution preferably those having a pH 
between 1 and 5. Suitable examples include acetic acid, succinic acid, 
glucuronic acid, cysteic acid, crotonic acid, itaconic acid, glutonic 
acid, formic acid, aspartic acid, adipic acid, and salts of any of these. 
Succinic acid, aspartic acid, adipic acid, and salts of acetic acid, e.g. 
sodium acetate are preferred. Also, the solubilization may also be carried 
out at a neutral pH by means of a chaotropic agent. Suitable agents 
include urea, sodium bromide, guanidine hydrochloride, KCNS, potassium 
iodide and potassium-bromide. Concentrations and volumes of such acid 
buffer or such chaotropic agent are as described in EP-PS No. 592,242. 
During or immediately after the supply of blood, the piston rod 8 is pushed 
so far into the interior of the device that the displaceable sleeve 52 of 
the capsule 45 is moved downwards into a sealing engagement in the through 
passage through the bottom wall 76 and to the second chamber 77. As a 
result, access is simultaneously opened to the biotin-batroxobin 
composition inside the uppermost chamber 80 of the capsule. 
When the device is ready for use, a blood sample is fed into the first 
chamber through a needle not shown and the hose 65 in a conventional 
manner, said blood sample preferably being admixed an anticoagulant also 
in a conventional manner. During the feeding of the blood through the hose 
65 and the opening 66 into the interior of the first chamber 70, air is 
removed from the chamber in a conventional manner. After the feeding of 
blood the hose 65 is removed, and the opening 66 is sealingly closed. 
Subsequently, the device with the blood is placed in a centrifuge which 
inter alia assists in sealingly compressing the various parts. The 
centrifuge causes the device to rotate about the axis of rotation 1. As a 
result of the centrifuging, the blood is separated in the first chamber 70 
into a plasma fraction settling radially inside the remaining portion of 
the blood, said remaining portion containing the red and the white blood 
cells. As described ink EP-PS No. 592,242 the platelets can be present in 
either fraction, as desired, by varying the speed and time of 
centrifugation. 
When the interface between the plasma and the remaining portion of the 
blood has stabilized, i.e. when the separation is complete, a reduction of 
the volume of the first chamber 70 is initiated by the piston rod 8 and 
consequently the piston 55 being pulled out. As a result, first a possible 
inner layer of air passes through the channels 4 and 21 into the second 
chamber 75, and a further moving of the piston 55 implies that also the 
plasma passes to the second chamber 75. The movement of the piston 55 is 
stopped when the entire layer of plasma has been forced into the second 
chamber 75, i.e. when the interface between the plasma fraction and the 
remaining portion of the blood has reached the inner wall 71 of the first 
chamber 70. 
In the second chamber 75, the plasma fraction comes into contact with the 
enzyme batroxobin with the result that fibrin monomer, which polymerizes 
immediately to a non-crosslinked fibrin polymer, is released from the 
plasma fraction. This process is performed while the device is being 
continuously centrifuged with the result that fibrin polymer is 
efficiently separated from the remaining portion of the plasma fraction, 
said fibrin polymer being formed by the reaction of the biotin-batroxobin 
composition and settling as a viscous layer along the cylindrical outer 
wall 72. When this separation has been completed, the centrifuging is 
stopped whereby the remaining relatively fluid portion of the plasma 
fraction can easily be pressed back into the first chamber 70 by the 
piston 55 first being raised for transferring air from the first chamber 
70 to the second chamber 75 followed by said piston 55 being pressed down. 
This transfer can be performed relatively easily and quickly before the 
viscous layer with fibrin polymer reaches the opening to the channel 21. 
Further measures can optionally be taken in order to prevent the viscous 
layer from reaching the inlet of the channel 21 too quickly, such as by 
providing a ring of upwardly projecting teeth 82 shown by dotted lines at 
the bottom 76. This centrifuging/draining procedure can be carried out two 
or more times, as may be required, to get as much of the plasma fluid out 
of the fibrin polymer as is possible. 
Once the remaining portion of the plasma fraction has been expelled from 
the second chamber 75, the displaceable sleeve 52 of the capsule 45 is 
further displaced downwards in such a manner that access is allowed to the 
lowermost chamber 81. At the same time or in connection with the latter 
displacement of the sleeve, the plug 49 of the syringe 58 is pressed 
completely downwards by means of a spindle acting from the outside in such 
a manner that the pH-4 buffer is transferred to the second chamber 75, 
which can be done while initiating a centrifugal agitation. The addition 
of the pH-4 buffer provides that fibrin polymer is dissolved therein, and 
the presence of the avidin-agarose composition in the lower chamber 81 
inside the capsule 45 provides that the biotin-batroxobin composition is 
bound in a conventional manner by the avidin. A continued displacement of 
the piston 55 causes the displaceable sleeve 52 on the capsule 45 to 
engage the partition means 36 and to a disengage the bottom wall 76 with 
the result that a free access is provided to the third chamber 77. As a 
result, the contents of the second chamber 75 can flow freely downwards 
into the third chamber 77. Preferably, the redissolving is carried out 
during centrifugal agitation which involves centrifugation and a series of 
stop-and-start of forward/reverse agitation motions. 
A continued centrifuging provides that the fibrin monomer solution can be 
separated in the third chamber through the annular filter unit 42 
retaining the relatively large particles of agarose and the batroxobin 
bound thereto via the biotin-avidin capture system. When the fibrin 
monomer solution has passed into the lowermost fourth chamber 78 as a 
result of the above centrifuging, said centrifuging is stopped and the 
fibrin-I-solution is easily transferred to the syringe 58 by a renewed 
retraction of the plug 59, the uppermost end of the length of pipe 46 of 
the capsule 45 engaging the length of pipe 47 forming the connection with 
the syringe 58. 
As fibrin polymer is separated from the plasma fraction in the second 
chamber 75 during a continued centrifuging and as the fibrin monomer 
solution is separated in the third chamber 77 by centrifuging it is 
possible to achieve a relatively high yield of fibrin I from the blood 
sample in question. 
The invention has been described with reference to a preferred embodiment. 
Many modifications can, however, be performed without thereby deviating 
from the scope of the invention. 
FIG. 2 illustrates examples of such modifications, as said FIG. 2 
illustrates a second embodiment of the invention which more or less 
corresponds to the embodiment of the invention shown in FIG. 1. The 
embodiment of FIG. 2 comprises a first chamber 90 and a second chamber 91 
separated by a piston 92, which comprises a hollow piston rod 93 defining 
the first chamber inwardly. Outwardly, the two chambers are defined by a 
portion of a substantially tubular member 94 forming and outer cylindrical 
wall 95 for the two chambers 90 and 91. Upwardly, the first chamber 90 is 
defined by a top wall 85 which in turn is formed by a top cover secured to 
the tubular member 94 by means of a ring 96 screwed into said tubular 
member 94. The top wall 85 defines a through opening for passage of the 
hollow piston rod 93. Downwardly, the second chamber 91 is defined by a 
bottom wall 96 formed by a circumferential inner flange in the tubular 
member 94. On the side adjacent the second chamber 91, the tubular member 
94 comprises a frusto-conical surface 97 inclining away from the piston 92 
towards the center of the second chamber 91. The bottom wall 96 defines a 
central through passage 98 to a third chamber 99. The third chamber 99 is 
defined by a partition means 100 and an annular filter unit 101 inserted 
between the bottom wall 96 and the partition means 100 and leading to a 
fourth annular chamber 102. The fourth chamber 102 is defined between a 
cup-shaped cover 103 secured to the tubular member 94 by threads. Said 
cover 103 retains through intermediary ribs 103 the partition means 100 in 
position centrally inside the tubular member 94 while squeezing the 
annular filter unit 101. 
A capsule 105 is secured on a centrally and upwardly projecting pin 104 on 
the partition means 100. The capsule 105 comprises a tubular portion 106 
with disk-shaped rings 107, 108 loosely attached thereto and defining 
chambers for the said enzymes indicated by the letters BB and AA, 
respectively, by means of a displaceably arranged sleeve. The disk-shaped 
rings are secured at the desired mutual distances on the length of pipe 
106 by means of shoulders shaped thereon by the outer periphery of the 
tubular member 106 being of a decreasing diameter from below and upwards. 
Through channels 115 and 116 are provided from the top of the first chamber 
90 to the bottom of the second chamber 91. These channels are provided by 
means of their respective fixed length of pipe 117 and 118, respectively, 
extending parallel to the axis of rotation of the device and being secured 
at the ends in associated openings in the top wall 95 and the bottom wall 
96. The channel connection between these lengths of pipe and the chambers, 
respectively, is provided by suitable bores and plugs secured therein. The 
lengths of pipe 117 and 118 extend through their respective opening in the 
piston 92. Sealing rings are provided everywhere so as to prevent leakage. 
A coupling 120 is secured centrally inside the piston 92 for coupling to a 
syringe 121 inside the hollow piston rod 93 and to the upper end of the 
pipe length 106 of the capsule 105. The coupling 120 carries a skirt 122 
projecting into the second chamber 91 and influencing the displaceable 
sleeve 110 on the capsule 105. As illustrated, the outer diameter of this 
sleeve 110 is adapted to the diameter of the through passage 98 downwards 
to the third chamber 99 in such a manner that the sleeve 110 is guided and 
retained by the bottom wall 96 in any position and consequently also in a 
lowermost position in which the sleeve 105 does not engage the lowermost 
disk-shaped ring 109 in the capsule and allows passage of fluid from the 
second chamber 91 downwards into the third chamber 99. A channel 123 
extends from the fourth chamber 102 and passes centrally upwards through 
the pin 104 on the partition means 100 and further upwards through the 
tubular member 106 of the capsule 105, whereby fluid is allowed to enter 
the syringe 121 therefrom. 
The device of FIG. 2 is used in completely the same manner as the device of 
FIG. 1, whereby means, of course, are also provided for coupling a hose 
thereto for the feeding of blood. 
The parts described forming part of the various devices are easily 
manufactured from suitable plastic materials by way of injection moulding, 
and the devices in question are therefore also relatively inexpensive and 
suited for disposable use. 
The invention has been described with reference to preferred embodiments of 
the device. The method according to the invention may, however, easily be 
conducted in a laboratory under aseptic conditions by means of a cup which 
can be closed by a lid. Plasma and enzyme is filled into the cup and by 
mixing and following centrifugation, the non-crosslinked fibrin polymer is 
separated onto the bottom or the wall of the cup as described above. After 
removing the remaining plasma fraction, the non-crosslinked fibrin polymer 
is redissolved by addition of a solvent and by way of centrifugal 
agitation as described above as well. 
EXAMPLE 
140 ml of whole blood and 20 ml of sodium nitrate anticoagulant (USP) was 
introduced into the first chamber 70 of the device described above. This 
combination was centrifuged for 2 minutes at about 6,000 RPM to provide a 
separation of plasma and blood cells. While continuing the centrifugation 
to main the separation, the piston was raised so as to transfer the 
innermost phase, i.e. the plasma, into the second chamber 75. 
Approximately 60 ml of plasma was transferred. This was treated with 30 
units of biotenylated batroxobin which was introduced into the second 
chamber 75 via the upper chamber 80 of the capsule 45 as described 
previously. The plasma and abroxobin were mixed at a lower speed, e.g. 
about 2,000 to 3,000 RPM and thereafter centrifuged for 9 minutes at 9,000 
RPM. 
The non-crosslinked fibrin polymer gel was precipitated as a thin gel layer 
onto the cylinder walls and the rotation was ceased. The remaining plasma 
fluid (serum) was then transferred back into the first chamber 70. This 
was followed by two further 1 minute centrifugations at 9,000 RPM to 
remove as much of the serum within the gel as possible. Following each 
such 1 minute centrifugation, the excess serum was transferred to the 
first chamber 70. 
Thereafter, a buffer solution comprising 3.5 ml of a 0.2M sodium acetate 
(pH 4.0) containing 24 mM calcium chloride was introduced into the second 
chamber 75 via the syringe 58. At this time, a centrifugal agitation 
consisting of 5-10 second spins at about 3,000 RPMs each in repeated 
forward/reverse cycles was carried out for 2 minutes to dissolve the 
fibrin polymer gel and provide a fibrin monomer-containing solution. To 
the so-prepared solution was added avidin agarose via the lower chamber 71 
of capsule 45. This was followed by a further centrifugal agitation 
consisting of 5-10 second spins at about 3,000 RPMs each in repeated 
forward/reverse cycles for 5 minutes. The resulting solution contained 
fibrin monomer plus a complex of avidin-agarose: biotin-batroxobin. 
This solution was transferred into the third chamber 77 and centrifugally 
filtrated through a 20 .mu.m annular Porex filter for 1 minute at 9,000 
RPM. The resulting fibrin monomer solution was collected into syringe 58 
as described previously. 
The so-formed fibrin monomer solution (fibrin I in this case) was 
repolymerized into a fibrin sealant by co-administration to a site in need 
of such a sealant with a 0.75M sodium carbonate/bicarbonate buffer at a 
ratio of fibrin I:buffer of 5:1.