CA2095623C - System and method for processing biological fluids - Google Patents

Abstract

A system for collecting and processing donated blood comprises a first porous medium interposed between a blood collection bag and a satellite bag and a second porous medium interposed between the blood collection bag and another satellite bag. The porous media are leucocyte depletion media. The system may also include one or more of the following: a red cell barrier medium, a separation medium, a gas inlet, and a gas outlet. The system can be used to centrifuge whole blood into one or more components, and includes a means for protecting the system during centrifugation.

Description

2cq56~3 SYSTE~ ~ND METHOD POR PROCESSING BIOI,OGICAL E~ CDS

This invention relates to a system for process-ing blood donated for the purpose of therapeutic transfusion of blood components and, particularly, ~0 to improved methods and apparatuses for preparing, from the donated whole blood, platelet-rich plasma (hereinafter PRP), packed red cells (hereinafter PRC), platelet concentrate (hereinafter PC~, and plasma. This invention also relates to a biological fluid processing system for processing biological fluid into its various components.

The development of plastic blood collection ~ags has facilitated the separation of donated whole blood into its various components and analogous products, thereby making these different blood products (e.g., platelet concentrates) av~ilAhle as a transfusion ~oduct.
With the p~CcAge of time and accumulation of research and cli~ical data, transfusion practices have changed greatly. One aspect of current practice is that whole blood is rarely administered;
rather, patients ne~ding red blood cells are given packed red cells, patients needing platelets are given platelet concentrate, and patients ne~ing D

- w~92J076~6 PCT/US91/08316 J
_ plasma are given plasma.
For this reason, the separation of blood into components has substantial therapeutic and monetary - -value. This is nowhere more evident than in treating the increased damage to a patient's immune system caused by the h;~h~r doses and sLLo~l~er drugs now used during chemotherapy for cancer patients.
These more aggressive chemotherapy protocols are directly implicated in the reduction of the platelet content of the blood to abnormally low levels;
associated internal and external bleeding additionally requires more frequent transfusions of PC, and this has put pressure on blood h~c to increase the platelet yield per unit of blood.
A typical component separation proce~e used in the United States, the citrate-phosphate-dextrose-adenine (CPDA-l) system, utilizes a series of steps to separate donated blood into three components, each comlole~ having substantial thela~eu~ic and monetary value. The ~ o~dule typically utilizes a blood collection bag which is - integrally attached via flexible tubing to at least one, and preferably two or more, satellite bags.
Using centrifugation, whole blood may be ceparated by differential sedimentation into such valuable blood components as plasma, packed red cells (PRC), platelets suspended in clear plasma (platelet-rich plasma, or PRP), platelet concentrate (PC), and ~lyo~le-ipitate (which may require extra processing).
A typical whole blood collection and processing proc l~e may include the following:
(1) A unit of donated whole blood (about 450 ml in United States practice) is collected from the 3S donor~s vein directly into the blood collection bag which contains the nutrient and anti-coagulant -i ~92/076~ PCT/US91/08316 209562~

cont~ining CPDA-l.

(2) The blood collection bag is centrifuged (slow speed, or "soft-spin" centrifugation) together with its satellite bags, thereby conce.l~.ating the S red cells as PRC in the lower portion of the blood collection bag and leaving in the upper portion of the bag a 5~c~ncion of PRP.

(3) The blood collection bag is trans~erred, with Qre not to di~L~ the interface between the ~ ..atant PRP layer and the ~ mented PRC layer, into a device known as a "plasma extractor." The plasma extractor or expressor typically includes front and back plates; the two plates are hinged together at their lower ends and spring hi~e~
lS toward each other such that a pressure of about 90 millimeters of me~ is developed within the bag.
With the blood collection bag positioned be-tween the two plates, a valve, seal or a closure in or on the flexible ~-lhing is ~rl~ allowing the ~u~el,latant PRP to flow into a first satellite bag.
As the PRP flows out of the blood collection bag, the interface with the PRC rises. In c~l~ellL
practice, the operator must closely observe the position of the interface as it rises and clamp off the c~n.~~ting tube when, in his judgment, as much PRP has been transferred as is possible, without allowing red cells to enter the first satellite bag.
This is a labor intensive and time ~-o.i ming G~el~ion during which the operator must visually monitor the bag and judiciously and arbitrarily ascertain when to shut-off the connecting tube.
The blood collection bag, now cont~;ning only PRC, may be detached and stored at 4~C until required for transfusion into a patient, or a valve or seal in the tubing may be opened so that the PRC
may be transferred to a satellite bag using either w~92/076~ pCT/US91/08316~

2~9~ 3 the pressure generated by the plasma extractor, or by placing the blood collection apparatus in a pressure cuff, or by elevation to obtain gravity flow.

(4) The PRP _o~ in;ng satellite bag, together with another satellite bag, is then removed from the extractor and centrifuged at an elevated G force (high speed or "hard-spin" centrifugation) with the time and speed adjusted so as to conce,.~late the platelets into the lower portion of the PRP bag.
When centrifugation is complete, the PRP bag contains sedimented platelets in its lower portion and clear plasma in its upper portion.

(5) The PRP bag is then placed in the plasma extractor, and most of the clear plasma is expressed into a satellite bag, leaving the PRP bag cont~ining only the c~imented platelets and about 50 ml of plasma; then in a s~lhsequent step, this platelet composition is dispersed to make platelet conc~nLLate (PC). The PRP bag, now containing a PC
~ u~L, is then detached and stored for up to five days at 20~-24~C, until needed for a transfusion of platelets. Multiple units of platelets (e.g., from 6-lO donors, if for transfusion into an adult patient) may be pooled into a single platelet transfusion.
(6) The plasma in the satellite bag may itself be transfused into a patient, or it may be separated by complex ~LG~e~ses into a variety of other valuable products.
Commonly used systems other than CPDA-l include Adsol, Nutricell, and SAG-N. In these latter systems, the collection bag contains only anti-coagulant, and the nutrient solution may be preplaced in a satellite bag. This nutrient solution is transferred into the PRC after the PRP

~092/076~ PCT~US91/08316 - 2~95623 has been separated from the PRC, thereby achieving a higher yield of plasma and longer storage life for the PRC.
In view of this, there is a growing need for an efficient system and method for separating a . biological fluid (e.g., whole blood) into its components. 8100d bank per~o~ne1 have responded to the increased need for blood components by attempt-ing to increase PRC and PC yields in a variety of ways. In separating the PRC and PRP fractions (e.g., step 3 above), blood bank per~nn~l have-attempted to express more PRP prior to stopping flow from the blood collection bag, but this has often ~o~ed to be count~L~loductive, since the PRP, and the PC c~hc~quently extracted from it, are frequently contaminated by red cells, giving a pink or red color to the normally light yellow PC. The pres~nc~ of red cell~ in PC is so highly undesirable that pink or red PC is frequently ~i~c~rded, or-subjected to recentrifugation, both of which in-crease operating costs and are labor intensive. As a result, blood bank per~oP~l must err on the side of caution by stopping the flow of PRP before it has been fully eXprecc~ Thus, the PC is ~l~o..Laminated, but the ~n~ se~ plasma, which is valuable, may be wasted.
This reflects another problem when attempting to increase the yield of individual blood componènts. While each comron~~t is valuable, any savings resulting from incre~;ng the yield may be offset by the increased labor cost, if the operator of the proc~csing system must continuously and carefully monitor the system to increase the yield.
The devices and methods of this invention alleviate the above-described problems and, in addition, provide a higher yield of superior quality WOg2/076~ PCT/US91/08316~
, 6 209~ 62 3 PRC and PC.
-The separation of the various blood components using centrifugation is attended by a number of other problems. For example, when PRP is S centrifuged to obtain a layer consisting principally of platelets concentrated at the bottom of the PRP-contAining bag, e.g., step 4 above, the platelets so conc~,.~ated tend to form a dense ay~e~te which must be dispersed in plasma to form platelet c~ ,.L~ate. The ~i~ro~sion step is ~ y carried out by gentle mixing, for example, by placing the bag on a moving table which rotates with a precessing tilted motion. This mixing requires several hours, a potentially undesirable delay, and lS is believed by many researchers to produce a partially ayyley~ted platelet concentrate. It is further believed that the platelets may be damaged by the forces applied during centrifugation.
Finally, a problem at~n~nt with the separation of various blood components using a multiple bag system and centrifugation is t'hat highly valuable blood components become ~la~ed in the conduits connecting the various bags and in the various devices that may be used in the system.
C~.ve~.~ional processing and storage te~-hnjques may also present probl~ms~ For example, air, in particular oxygen, ~-~nt in stored blood and blood components, or in the storage contAin~ may lead to an impairment of the quality of the blood components, and may decrease their storage life.
More particularly, oxygen may be A~COç~ Ated with an increased metabolic rate (during glycolysis), which may lead to decre~se~ storage life, and decreased viability and function of whole blood cells. For example, during storage red blood cells metabolize glucose, producing lactic and ~y~uvic acids. These 2~5623 acids decrease the pH of the medium, which in turn decreases metabolic functions. Furthermore, the presence of air or gas in the satellite bag may present a risk when a patient is transfused with a blood component. For example, as little as 5 ml of air or gas may cause severe injury or death.
Despite the deleterious effect of oxygen on storage life and the quality of blood and blood components, the prior art has not addressed the need to remove gases from blood ~Lo~essing systems during collection and processing.
In addition to the above-listed components, whole blood contains white blood cells (known collectively as leucocytes) of various types, of which the most important are granulocytes and lymphocytes. White blood cells provide protection against bacterial and viral infection. The transfusion of blood components which have not been - leucocyte-depleted is not without risk to the patient receiving the transfusion. Some of these risks are detailed in U.S. Patent 4,923,620, and in U.S. Patent 4,880,548.

In the above described centrifugal method for separating blood into the three basic fractions, the leucocytes are present in ~ubstantial quantities in both the packed red cells and platelet-rich plasma fractions. It is now generally àccepted that it is highly desirable to reduce the leucocyte concentration of these blood components to as low a - level as possible. While there is no firm criterion, it is generally accepted that many of the undesirable effects of transfusion would be reduced if the leucocyte content were reduced by a factor of about loO or more prior to administration to the patient. This approximates reducing the average W0~2~076s6 PCT/US91/08316-, 2(~5623 total content of leucocytes in a single unit of PRC
-to less than about 1 x lo6, and in a unit of PRP or PC to less than about 1 x 105. Devices which have previously been developed in attempts to meet this objective have been based on the use of packed fibers, and have generally been referred to as filters. However, it would appear that proa~ c utilizing filtration hA~e~ on separation by size cannot ~v~ae~d for two re~o~C. First, leucocytes can be larger than about 15 ~m (e.g., granulocytes and macrocytes) to as small as 5 to 7 ~m (e.g.,' lymphocytes). Together, granulocytes and lymphocytes represent the major p~o~o~Lion of all of the leucocytes in normal blood. Red blood cells are about 7 ~m in diameter, i.e., they are about the same size as lymphocytes, one of the two major cl~c~e~ of leucocytes which must be removed.
.S~co~ly, all of these cellc deform so that they are able to pass through much smaller ~ponin~s than their normal ~ize. Acco~dingly, it has been widely accepted that removal of leucocytes is accamplished mainly by adsorption on the internal surfaces of porous media, rather than by filtration.
Leucocyte depletion i5 particularly important with respect to a blood comronent such as PC.
Platelet ao~c~ntrates prepared by the differential centrifugation of ~lood components will have varying levels of leucocyte contamination related to the time and to the magnitude of the force"developed during centrifugation. The level of leucocyte contamination in unfiltered conventional platelet preparations of 6 to 10 pooled units is generally at a level of about 5 x 108 or greater. It has been demonstrated that leucocyte removal efficiencies of 81 to 85~ are sufficient to reduce the incidence of febrile reactions to platelet transfusions. Several ~092/076~6 PCT/US9l/08316 .
~09~23 other recent studies report a reduction in alloim~unization and platelet refractoriness at levels of leucocyte contamination below about 1 x 107 per unit. For a single unit of PC averaging a leucocyte contamination level (under current practice) of about ~ x 107 leucocytes, the goal after filtration is less than 1 x 106 leucocytes. The existing studies, therefore, suggest the desirability of at least a two log (99%) reduction of leucocyte contamination. More le~l~ studies suggest that a three log ~99.9~) or even a four log (99.99~) reduction would be significantly more beneficial.
An additional desirable criterion is to ~e~ict platelet loss to about 15~ or less of the original platelet concentration. Platelets are notorious for being "sticky", an expression reflecting the tendency of platelets ~~-crende~ in blOoa plasma to aahere to any non-physiological surface to which they are ~Yp~fO~. Under many circ~mstances, they also adhere ~ yly to each other.
In any system which depends upon filtration to remove leucocytes from a platelet s~-crencion~ there will be substantial contact between platelets and the internal surfaces of the filter assembly. The filter assembly must be such that the platelets have minimal adhesion to, and are not significantly adver~ely affected by contact with, the filter assembly~s internal surfaces.
If the leucocyte depletion device comprises a porous structure, microaggregates, gels, fibrin, fibrinogen and fat globules tend to collect on or within the pores, causing blockage ~hich in~ihits flow. Conventional processes, in which the filter for depleting leucocytes from PRC is pre-conditioned - ' W~92/076~ PCT/US91/08316' 209~623 by passing saline through the filter assembly with or without a post-filtration s~l;n~ flush, are undesirable because the liquid content of the transfusion is unduly increased, thus potentially overloading the patient's circulatory system with liquid. An objective of an embodiment of this -invention i5 a leucocyte depletion device which removes le~oG~Les and these other elements with high efficiency and without clogging, requires no ~L~G..litioning prior to ~lv~e_sing PRC derived from f~ ly drawn blood, and does not require post-filtration flushing to reclaim red cells remaining in the filter.
Because of the high cost and limited availability of blood components, a device comprising a porous medium used to deplete leucocytes from biological fluid r~ deliver the highest possible proportion of the component ~le_cnt in the donated blood. An ideal device for the leucocyte depletion of PRC or PRP would be ;lr~ ive, relatively ~mall, and be capable of rapidly ~ro~e_sing blood components obt~inP~ from about one unit or more of biological fluid (e.g., donated whole blood), in, for example, less than about one hour. Ideally, this device would reduce the leucocyte content to the lowest possible level, while maximizing the yield of a valuable blood com~o,.~,L while minimizing an ~Yre~cive~
~orhi~ticated, labor intensive effort by t~e operator of the system. The yield of the blood component s~o~ld be maximized while at the same time delivering a viable and physiologically active component--e.g., by minimizing damage due to centrifugation, and/or the presence of air or gas.
It may also be preferable that the PRC porous medium be capable of removing platelets, as well as ~092/076~ PCT/US91~08316 .

20g5623 fibrinogen, fibrin strands, tiny fat globules, and other components such as microaggregates which may be present in whole blood.

Definitions ?he following definitions are used in refe~ e to the invention:
(A) Blood Product or Biological Fluid: anti-coagulated whole blood (AWB); packed red cells obtained from ANB; platelet-rich plasma (PRP) obtained from AWB; platelet ~o~ ate (PC) obtAin~ from AWB or PRP; plasma obtained from AWB
or PRP; rea cells separated from plasma and re~ e.~led in physiological fluid; and platelets separated from plasma and r~s~r~nAed in ~5 physiological fluid. Blood ~lGd~ct or biological fluid also includes any treated or untreated fluid ~R~QCiAted with living organicms~ particularly blood, including whole blood, warm or cold blood, and stored or fresh blood; treated blood, such as blood diluted with a physiological solution, including but not limited to s~line, nutrient, and/or anticoagulant solutions; one or more blood components, such as platelet conce~.L~Le (PC), platelet-rich plasma (PRP), platelet-free plasma, platelet-poor plasma, plasma, or packed red cells (PRC); analogous blood ~Gd~cts derived from blood or a blood com~o"e"L or derived from bone marrow.
The biological fluid may include leucocytes, or may be treated to remove leu~yLes. As used herein, blood product or biological fluid refers to the components described above, and to similar blood products or biological fluids obtained by other means and with similar properties. In accordance with the invention, each of these blood products or biological fluids is processed in the manner I W~92/076~ PCT/US91/0831~

209~1;23 described herein.
(B) Unit of Whole Blood: Blood banks in the United States commonly draw about 450 milliliters (ml) of blood from the donor into a bag which contains an anticoagulant to prevent the blood from clotting. However, the amount drawn differs from patient to patient and donation to donation. Herein the quantity drawn during such a donation is defined as a unit of whole blood.
(C) Unit of Packed Red Cells (PRC), Platelet-rich Plasma (PRP) or Platelet Con~entrate (PC): As used herein, a "unit" is defined by the United States~ practice, and a unit of PRC, PRP, PC, or of red cells or platelets in physiological fluid or plasma, is the quantity derived from one unit of whole blood. It may also refer to the quantity drawn during a single donation. Typically, the volume of a unit varies. For example, the volume of a unit of PRC varies considerably ~pen~;ng on the hematocrit (percent by volume of red cell~) of the drawn whole blood, which is ~ lly in the range of about 37% to about 54%. The concomitant hematocrit of PRC, which varies over the range from about 50%
to over 80%, depends in part on whether the yield of one or another blood ~Lod~ct is to be minimized.
Most PRC units are in the range of about 170 to about 3~0 ml, but variation below and above these figures is not uncommon. Multiple units of some ~loc~ components, particularly platelets, may be pooled or combined, typically by combining 6 or more units.
(D) Plasma-Depleted Fluid: A plasma-depleted fluid refers to any biological fluid which has had some quantity of plasma removed therefrom, e.g., the platelet-rich fluid obtained when plasma is separated from PRP, or the fluid which remains after ~Q92/076~ PCT/US91/08316 plasma is removed from whole blood.
(E) Porous medium: refers to the porous medium through which one or more blood components or biological fluids pass. The PRC porous medium depletes leucocytes from the packed red cell component. The platelet or PRP porous medium refers generically to any one of the media which deplete leucocytes from the non-PRC blood compQ~ts, i.e., from PRP or from PC. The red cell barrier medium -blocks the passage of red cells and depletesleucocytes from PRP to a greater or lesser degree while allowing the p~Age of platelets.
As noted in more detail below, the ~o~ous medium for use with PRC may be formed from any natural or synthetic fiber (or from other materials of similar surface area and pore size) compatible with blood. The porous medium may remain untreated.
Preferably, the critical wetting surface tension (CWST) of the ~OLO~S medium is within a certain range, as noted below and as dictated by its int~n~e~ use. The pore surfaces of the medium may be modified or treated in order to achieve the desired CWST. For example, the CWST of a PRC porous medium is typically above about 53 dynes/cm.
2S The ~o~ous medium for use with PRP may be formed from any natural or synthetic fiber or other porous material compatible with blood. The ~o~us medium may remain unL~eated. Preferably, the CWST
and zeta potential of the ~o~ous medium are within certain ranges, as disclos~ below and as dictated by its intended use. For example, the CWST of a PRP
porous medium is typically above about ~0 dynes/cm.
The porous media according to the invention may be connected to a conduit interposed between the containers, and may be positioned in a housing which in turn can be connected to the conduit. As used ~'0 92/076~6 PCI/US91/08316' ._ , herein, filter assembly refers to the porous medium positioned in a suitable housing. An exemplary filter assembly may include a leucocyte depletion ass~m~ly or device or a red cell barrier assembly or device. A biological fluid ~o~essing system, such as a blood collection and processing system, may comprise porous media, preferably as filter assemblies. Preferably, the porous medium forms an interference fit at its edges when assembled into the housing.
The porous medium may be configured as a flat - sheet, a corrugated sheet, a web, or a membrane.
The porous medium may be pre-formed, and configured as hollow fibers, although it is not intended that fS the invention should be limited thereby.
(F) Separation Medium: A separation medium refers to a porous medium effective for separating one component of a biological fluid from another component. The separation media according to the invention are suitable for passing at least one component of the blood ~ ~ct or biological fluid, particularly plasma, therethrough, but not other components of the blood product or biological fluid, particularly platelets and/or red cells.
-25 As noted in more detail below, the separation medium for use with a biological fluid may be formed from any natural or synthetic fiber or from a ~o.o~s or permeable membrane (or from other materials of similar surface area and pore size~ compati~le with a biological fluid. The surface of the fibers or membrane may be unmodified or may be modified to achieve a desired property. Although the separation medium may remain ~ eated, the fibers or membrane are preferably treated to make them even more effective for separating one component of a biological fluid, e.g., plasma, from other '- 239~623 components of a biological fluid, e.g., platelets or red ~ . The separation medium is preferably treated in order to reduce or eliminate platelet adherence to the medium. Any treatment which reduces or eliminates platelet adhesion is included within the scope of the present invention.
Furthermore, the medium may be surface modified as disclosed in U.S. Patent 4,880,548, in order to increase the critical wetting surface tension (CWST) lo of the medium and to be less adherent of platelets.
Defined in terms of CWST, a preferred range of CWST
for a separation medium according to the invention is above about 70 dynes/cm, more preferably above about 90 dyneslcm. Also, the medium may be subjected to gas plasma treatment in order to reduce platelet adhesion. Preferably, the critical wetting - ~urface tension (CWST) of the separation medium is within a certain range, as noted below and as dictated by its intended use. ~he pore surfaces of the medium may be modified or treated in order to achieve the desired CWST.
The separation medium may be pre-formed, multi-layered, and/or may be treated to modify the surface of the medium. If a fibrous medium is used, the fibers may be treated either before or after forming the fibrous lay-up. It i~ preferred to modify the fiber ~urfaces before forming the fibrous lay-up because a more cohesive, ~on~er ~d~ct is obtained after hot compression to form an integral filter element. The eparation medium is preferably pre-formed.
~ he separation medium may be configured in any suitable fashion, such as a flat sheet, a corrugated cheet, a web, hollow fibers, or a membrane.
(G) Voids volume is the total volume of all of ~g2/076~6 PCT/US9l/08316' the pores within a porous medium. Voids volume is - expressed hereinafter as a pe~cell~age of the apparent volume of the porous medium.
(H) Measurement of fiber surface area and of average fiber diameter: In accordance with the invention, a useful technique for the measurement of fiber surface area, for example by gas adsorption, is generally referred to as the "BETU measurement.
The surface area of melt blown webs can be used to calculate average fiber diameter, using PBT as an example: -~

Total volume of fiber -in 1 gram = l8 cc (where 1.38 = fiber density of PBT, g/cc) hence ~4d2L 1.38 (1) Area of the fiber is ~dL = Af (2) Dividing ~1) by (2), d = 1 31A

and d = 1 38Af if where L = total length in cm of 1 gram of fiber, d = average fiber diameter in centimeters, and Af = fiber surface area in cmZ/g.
If the units of d are micrometers, the units of Af become M2/g (~quare meters/gram), which will be used hereinafter.
(I) Critical Wetting Surface Tension: As disclosed in U.S. Patent 4,880,548, ~he CWST of a porous medium may be determined by individually applying to its surface a series of liquids with ~ W092/076~ PCT/US91/08316 209~623 surface tensions varying by 2 to 4 dynes/cm and observing the absorption or non-absorption of each liguid over time. The CWST of a porous medium, in units of dynes/cm, is defined as the mean value of S the surface tension of the liquid which is absorbed and that of the liquid of nei~hhoring surface tension which is not absorbed within a predetermined amount of time. The absorbed and non-absorbed values ~erenA princir~lly on the surface characteristics of the material from which the porous medium is made and ~~~on~rily on the pore size characteristics of the ~OlvUS medium.
Liquids with surface tensions lower than the CWST of a porous medium will ~ n~o~cly wet the medium on contact, and, if the pores of the medium are interconnected, liquid will flow through the medium readily. Liquids with surface tensions higher than the CWST of the p~v~s medium may not flow at all at low differential pres~uue-~ or may flow unevenly at sufficiently high differential ~ uu~es to force the liquid through the ~OL~US
medium. In order to achieve adequate priming of a fibrous medium with a li~uid such as blood, the fibrous medium preferably has a CWST in the range of about 53 dynes/cm or higher.
For the porous medium which is used to ~lo~ess PRC, it is preferred that the CWST be held within a range somewhat above the CWST of ~l~leated polyester fiber (52 dynes/cm), for example, above about 53 dynes/cm, more preferably, above about 60 dynes/cm.
For the porous medium which is used to proc~Cc PRP, it is preferred that the CWST be held within a range abo~e a~out 70 dynes/cm.
(J) General procedure for measuring zeta potential: Zeta potential was measured using a sample cut from a ~ inch thick stack of webs.

~-~92~076~6 PCT~US9l/08316 20955~3 The zeta potential was measured by placing the sample in an acrylic filter holder which held the sample snugly between two platinum wire screens 100 x 100 mesh (i.e., 100 wires in each direction per inch). The meshes were connected, using copper .wire, to the terminals of a Triplett Corporation model 3360 Volt-Ohm Meter, the mesh on the u~,eam side of the sample being connected to the positive terminal of the meter. A ~I ~ffered solution was lo flowed through the sample using a differential pres-sure of 45 inches of water column across the filter holder and the effluent was collected. For measurements at pH 7, a buffered so~lution was made by adding 6 ml pH 7 buffer ~Fisher Scientific Co.
-catalog number SB108-500) and 5 ml pH 7.4 buffer (Fisher Scientific Co. catalog number SBllO-500) to 1 liter ~y~oyen-free deionized water. For measure-ments at pH 9, a buffered ~olution was made by add-ing 6 ml pH 9 buffer (Fisher Scientific Co. catalog number SB114-500) and 2 ml pH 10 buffer (Fisher Scientific Co. catalog number SB116-500) to 1 liter pyrogen-free deionized water. m e electrical poten-tial across the filter holder was measured during flow (it required about 30 seconds of flow for the potential to stabilize) and was ~oLIected for cell polarization ~y subtracting from it the electrical potential measured when flow was stopped. During the period of flow the pH of the liquid was measured using a Cole-Parmer model J-5994-10 p~ meter fitted with an in-line model J-5993-90 pH probe. The con-ductivity of the liquid was measured using a Cole-Parmer model J-1481-60 conductivity meter fitted with a model J-1481-66 conductivity flow cell. Then the polarity of the volt meter was reversed, and the effluent was flowed backwards through the filter holder using a differential pressure of 45 inches of ~092/076~6 PCT/US91/08316 .

water column. As in the first instance the electri-cal potential measured during flow was corrected for cell polarization by subtracting from it the elec-trical potential measured after flow was stopped.
The average of the two corrected potentials was taken as the streaming potential.
The zeta potential of the medium was derived from the streaming potential using the following relatio~;r (J. T. ~avis et al., Interfacial Phenomena, Academic Press, New York, 1963~:
Zeta Potential = 4DP ~ EsA

where n is the viscosity of the flowing solution, D
is its diele~L~-ic ~o..~Lant, A is its conductivity, Es is the streaming potential and P is the ~le~sule drop across the sample during the period of flow.
In these tests the quantity 4 ~D/DP was equal to 0.800.
(K) Tangential flow filtration: As used herein, tangential flow filtration refers to passing or circulating a biological fluid in a generally parallel or tangential manner to the surface of the separation medium.

Summary of the Invention In the devices and methods of this invention, leucocyte depletion of a biological fluid (e.g., PRC
or PRP~ is carried out at the time of ~lGcessing, which, in the United States, is generally within about 6 to 8 hours of the time the blood is drawn.
Thus, as a biological fluid is transferred from the bag in which it is contained, leucocytes are removed by the ~Lo~Liate porous medium, and leucocyte-depleted biological fluid is collected in the satellite bag. In accordance with the invention, a system is provided whereby a biological fluid such wo~s2/076~ PCT/US91/08316 2095~23 as whole blood is processed to form PRP and PRC.
PRP is leucocyte depleted by interposing between the blood collection bag and a first satellite bag at least one porous medium for depleting leucocytes S from PRP; PRC is leucocyte depleted by interposing between the blood collection bag and a ~ocon~
satellite bag at least one porous medium for removing leucocytes from PRC.
The invention also comprises a ~elll.ifugation system wherein one (or both) of the inte~
leucocyte depletion filter assemblies is (are) cooperatively arranged with a centrifuge bucket in a manner such that the filter~assembly, the porous medium in the filter assembly, and the blood bags are not damaged by the very large forces generated during the cell~,ifugation process.
P,oce_~es and systems according to the invention may also include a red cell barrier medium that allows the passage of one component of the biological fluid, but ~-eve..Ls the p~c~ge of another component through the medium, there~y eliminating the need for continuous monitoring by an operator and increasing the efficiency with which a biological fluid such as whole blood is separated 2S into one or more components.
Additionally, pro~eC~ and systems according to the in~ention may include a gas outlet that allows gas that may be ~le_cnt in the system out of the system.
P-o~ ?~ and systems according to the invention may also include a gas inlet that allows gas into the system to recover a biological fluid that may ~e entrapped or retained during processing.
The invention also involves the treatment of a ~iological fluid to non-centrifugally separate at least one component from the biological fluid, e.g., W092/076~ PCT/US91/08316 treating PRP to obtain plasma and PC, or separating plasma from whole blood. Proc~se~ and devices according to the invention utilize a separation medium that allows the passage of one comron~nt of S the biological fluid, such as plasma, but prevents r~s~ge of other components, such as platelets or red cells, through the medium, thereby eliminating the need for ~hard-spin~ centrifugation as a ~LC~ing step. Tangential flow of a biological fluid parallel to the u~LLeam surface of the se~aLing medium permits the passage of plasma tL~l. the medium, while reducing the tendency for Ar components or platelets to adhere to the surface of the medium, thus assisting in the ~e~el~ion of p~ ge of platelets through the separation medium. The hydrodynamics of flow - parallel to a sur~ace are indeed believed to be such that during flow parallel to the surface, platelets develop a ~pin which causes them to be recovered from the surface.

Brief ~escription of the Drawinqs Figure 1 is an embodiment of a biological fluid ~lO~ in~ system according to the invention, whereby biological fluid is separated into components by centrifugal separation.
~ igure 2 is another embodiment of a biological fluid ~o-~-cing ~ystem according to the invention, including a non-centrifugal separation device.
~ igure 3 is an emh~iment of the invention which inooL~ol~tes a gas inlet and a gas outlet.
Figure 4 is an exploded perspective view of one emhoAiment of a filter assem~ly, a centrifuge bucket, and a holder to properly position the filter assembly on the bucket.
Figure 5 is an elevation of an embodiment of 20~5623 .

the present invention.
Figure 6 is a cross-section of an embodiment of the invention, showing the first fluid ~low path in a separation device according to the invention.
Figure 7 is a section of Figure 6, along 7--7 Figure 8 is a section of ~igure 6, along 8--8.
Figure 9 is a cross-section of an embodiment of the invention, showing the second fluid flow path in a separation device according to the invention.
Figure 10 is a section of Figure 9, along 10--10.
Figure 11 is a section of Figure 9, alongll--11.

S~ecific DescriPtion of the Invention The present invention involves a biological fluid, preferably blood, collection and processing assembly comprising a first container and a second cont~i n~r, and a conduit interconnecting the first cont~iner with the secon~ container; and at least one third container and a conduit inte~o~ ecting the first container with the third container; and having interposed between the first container and a second container, at least one first porous medium;
and having interposed between the first container and a third container, at least one second porous medium. The first porous medium may be a leucocyte depletion medium, a red cell barrier medium, an assembly comprising a leucocyte depletion medium and a red cell barrier medium, or combinations thereof.
The ~con~ porous medium may be a leucocyte depletion medium which may, optionally, include a microag~leyate filter element and/or a gel pre-filter element. As shown in more detail below, the assembly may also include additional containers, porous media, and conduits interconnecting the containers and porous media.
In another embodiment of the invention, the W092/0~6~ PCT/USgl/083t6 209a~23 blood collection and processing assembly comprises containers interconnected with a con~l7it~ and a porous medium interposed in the CQ~Allit for depleting leucocytes from PRC wherein the porous S medium has a CWST greater thAn about 53 dynes/cm.
In another embodiment of the invention, the blood collection and processing assembly comprises containers interconnected with a ~ond~it~ and a porous medium interposed in the conduit for depleting leucocytes from PRP wherein the porous medium has a CWST greater than about 70 dynes/cm.
The invention also involves a biological fluid ~o~ssing system comprising a first contAin~; a fir~t porous medium comprising a red cell barrier medium communicating with the first con~Ainer~ and defining a'-first flow path; and a s~con~ porous medium comprising a lel~oçyte depletion medium communicating with the first con~inor~ and defining a ~oc~nA flow path. As shown in more detail below, the system may also include additi~n~l con~Ainorc, flow paths, and ~O~GUS media.
The i~lvel~Lion also involves a method for collecting and pro~-ccing blood comprising collecting whole blood in a con~Ai~r; ~e.-~.ifuging the whole ~lood; passing the ~ atant layer of the ~I.LLifuged blood th~o~h a first porous medium, the first ~VLGUS medium comprising at least one of a leucocyte depletion medium, a red cell barrier medium, and a combined lellco~yte depletion red cell barrier medium; and passing the sediment layer of the C~ ifuged blood t~rough a ~o~ t~s medium, the ~c~ porous medium comprising a le~cocyte depletion medium.
The inv~l~ion also involves a method for processing a ~iological fluid comprising expressing a biological fluid from a first container to a first W0~2/076~ PCT/US91/08316 209~623 porous medium comprising a red cell barrier medium;
and e~le~sing a biological fluid from the first container to a ~?COnA ~GLv~ medium. As shown in more detail below, the method may also include ~,ocessing the fluid through additional containers, flow paths, and porous media.
An exemplary biological fluid collection and ~ocessing system is shown in Figure 1. The biological fluid processing system is generally lo denoted as lo. It may comprise a first container or collection bag 11; a needle or cannula 1 adapted to be inserted into the donor; an optional red cell barrier assembly 12; a first leucocyte depletion assembly 13; a ~ecQn~ con~Ain-~ (first satellite bag) 41; an optional fourth container (third satellite bag) 42; a second leucocyte depletion assembly 17; and a third container (second satellite bag) 18. Each of the assemblies or containers may be in fluid communication ~ Uy}l tubing, preferably flexible tubing, 20, 21, 25, 26, 27 or 28. The first leucocyte depletion assembly preferab~y includes a ~o,v~s medium for passing PRP; the second cocyte depletion assembly preferably includes a ~u,ou~ medium suitable for passing PRC. A seal, valve, clamp, or transfer leg closure or cannula ~not illu~Ll~Led) may al~o be positioned in or on the tubing or in the collection and/or satellite bags. The seal (or ~e~l~) is v~"el when fluid is to be transferred between bags.
In another exemplary configuration, the blood ~LG~essing system shown in Figure 2 is the same as the exemplary system shown in Figure 1, except that the portion of the system downstream of leucocyte depletion assembly 13 includes a separation assembly 14, preferably a non-centrifugal separation assembly.

wos2/o76s6 PCT/US91~08316 .

2~
In another exemplary configuration, shown in Figure 3, the invention may also comprise at least one gas inlet Sl, 53 and/or at least one gas outlet 52, 54. The system of Figure 3 includes a first 5 contA i n~r or collection bag ll in fluid communication with an optional red cell barrier assembly 12, gas inlet 53, a lel~coçyte depletion-assembly 13, and gas outlet 54. First con~ r ll may also be in fluid communication with a gas inlet 51, a leucG~y~e depletion assembly 17, and a gas outlet 52. As shown in more-detail below, the assembly may also include additional containers, flow paths, and ~o~v~s media.
Any number and combinations of assemblies, 15 porous media, containers, and conduits are suitable.
One skilled in the art will reco~i7e that the invention as described here may be ~ec~l,figured into different combinations, which are included within the scope of the invention.
Each of the components of the assembly will now be described in more detail below.
The containers which are used in the biological fluid ~oo~ ~ing assembly may ~e constructed of any material compatible with a biological fluid, such as whole blood or a blood component, and capable of withs~n~inq a ~el.L ifugation and sterilization environment. A wide variety of these containers are already known in the art. For example, blood collection and satellite bags are typically made from plasticized polyvinyl chloride, e.g. PVC
plasticized with dioctylphthalate, diethylhexylphthalate, or trioctyltrimellitate. The bags may also be formed from polyolefin, polyurethane, polyester, and polycarbonate.
3S As used herein, the tubing may be any conduit or means which provides fluid communication between w~92/076~ PCT/US91/08316 209~623 the containers, and is typically made from the same flexible material as is used for the containers, preferably plasticized PVC. m e tubing may extend into the interior of the container, and may be used as a siphon, for example. There may be a number of tubes providing fluid communication to any individual con~ine~ and the tubes may be oriented in a number of ways. For example, there may be at least two tubes oriented at the top of the collection bag, or at the bottom of the bag, or a tube at each end of the bag.
~ dditionally, the tubes, assemblies, porous media, and cont~in~rs~ may be oriented to define different flow paths. For example, when whole blood is ~lo~sse~, the PRP may flow along a first flow path, e.g., through the red cell barrier assembly (if present~, a PRP l~rocyte depletion assembly, and into a satellite bag (e.g., a ~DCon~ cont~in~).
Similarly, the PRC may flow along a ~?con~ flow path, e.g., through the PRC leucocyte depletion assembly, and into a satellite bag (e.g., a third con~inDr). Since independent flow paths may be present, biological fluids (e.g., PRP and PRC) may flow con~ e"~ly, or sequentially.
A seal, valve, clamp, transfer leg closure, or the like is typically located in or on the tubing.
It is intended that the ~escnt invention is not limited by the type of material used to construct the containers or the çon~l~it which connects the containers.
The composition of the various porous media will ~epen~ in part on the function desired, e.g., red blood cell blockage or leucocyte depletion. A
preferred composition of the various porous media is 35- a mat or web composed of fibers, which are preferably thermoplastic. The fibers of the porous W0~2/07656 PCT/US91/08316 . , 2~9~623 media may comprise any fiber compatible with biological fluid, and may be either natural or synthetic fibers.- In accordance with the invention, the fibers are preferably treated or modified in S order to achieve or increase the CWST. For example, the fibers may be surface modified to increase the critical wetting surface tension (CWST) of the fibers. For example, the treated or ~,~eated fibers used in the PRC porous medium preferably have a CWST above about 53 dynes/cm; for the PRP ~ ~us medium, above about 70 dynes/cm. Also, the fibers may be bonded, fused, or otherwise fixed to one another, or they may be merh~nically entwined.
Other porous media, for example, open cell foamed plastics, surface modified as noted above, may be similarly used.
While the porous media can be ~oduced from any material compatible with biological fluid, practical considerations dictate that consideration be given first to the use of commercially av~ hle materials. The ~lOUS media of this i~l~.Lion may be preferably formed, for example, from any synthetic polymer c~pAhle of forming fibers and of serving as a substrate for grafting. Preferably, the polymer ~ be c~pAhle of reacting with at least one ethylenically unsaturated monomer under the influence of ionizing radiation without the matrix being significantly or excessively adversely affected by the radiation. Suitable polymers for use as the substrate include, but are not limited to, polyolefins, polyesters, polyamides, polysul-fones, acrylics, polyacrylonitriles, polyarA~ides, polyarylene oxides and sulfides, and polymers and copolymers made from halogenated olefins and un-saturated nitriles. Examples include, but are notlimited to, polyvinylidene fluoride, polyethylene, ~92/076s6 PCT/US91/08316 _ polypropylene, cellulose acetate, and Nylon 6 and 66. Preferred polymers are polyolefins, polyesters, and polyamides. The most preferred polymer is poly-butylene terephthalate (PBT).
Surface characteristics of a fiber may remain unmodified, or can be modified by a number of methods, for example, by chemical reaction including wet or dry oxidation; by coating the surface by depositing a polymer thereon; or by grafting reactions wherein the substrate or fiber surface is activated prior to or during wetting of the fiber surface by a monomer solution by e~o~ule to an ~ source such as heat, a Van der Graff generator, ultraviolet light, or to various other forms of radiation; or by subjecting the fibers to gas plasma treatment. A preferred method is a - grafting reaction using gamma-radiation, for ex-ample, from a cobalt source.
An exemplary radiation grafting ~echnique em-ploys at least one of a variety of monomers each comprising an ethylene or acrylic moiety and a second group, which is preferred to be a hyd~o~hilic group (e.g., -COOH, or -OH). Grafting of the fibrous medium may also be accomplished by compounds containing an ethylenically unsaturated group, such as an acrylic moiety, combined with a hydlo~yl group, preferably monomers such as llydlo~ethyl methacrylate (HENA), or acrylic acid. The compounds con~ an ethylenically unsaturated group may be combined with a ~con~ monomer such as methyl acrylate (MA~, methyl methacrylate (MMA), or methacrylic acid (MAA~. MA or MNA are preferably incorporated into the porous medium used to treat PRC, and MAA is preferably incorporated into the 3S porous medium used to treat the PRP. Preferably, the MAA to HEMA monomer weight ratio in the . ~0~2/076~ PCT/US91/08316 209~3 modifying mixture may be between about .ol:1 and about 0.5:1; preferably, the MA or MMA to HENA
monomer weight ratio in the modifying mixture may be between about .ol:l and about 0.4:1. Use of HEMA
contributes to a very high CWST. Analogues with similar functional characteristics may also be used to modify the surface characteristics of fibers.
It has been observed that porous media, surface treated using some grafting monomers or combinations lo of monomers, behave differently with respect to the span between the surface tension of the liquid which is absorbed and the surface tension of the liquid which is not absorbed when determin~ng the CWST.
This span can vary from less than 3 to as much as 20 or more dynes/cm. Preferably, the media has a span between the absorbed and non-absorbed values of about 5 or fewer dynes/cm. This choice reflects the greater precision with which the CWST can be ~ Lolled when narrower s~ns are selected, ~lhe;t media with wider spans may also be used. The use of the narrower span is preferred in order to improve product quality ~oll~.ol.
Radiation grafting may increase fiber-to-fiber ho~in~ in a fibrous medium. Consequently, a fibrous medium which ~Yhihit5 little or no fiber-to-fiber hon~ing in an u.lL,eated state may exhibit significant fiber-to-fiber ho~ing after the fibers have been radiation grafted to increase the CWST of the medium.
For the ~o~us media for use with a biological fluid such as PRP, a preferred range for the CWST of the fiber is preferably above about 70 dynes/cm, typically about 70 to 115 dynes/cm; a more preferred range is 90 to ioo dynes/cm, and a still more pre-ferred range is 93 to 97 dynes/cm. A preferred range for the zeta potential (at the pH of plasma W~92/076~ PCT/US9l/08316 209~6~3 (7.3)~ is about -3 to about -30 millivolts, a more preferred range is about -7 to about -20 millivolts, and a still more preferred range is about -10 to about -14 millivolts.
In packed red cells, as well as in whole blood, the red cells are suspended in blood plasma, which has a surface tension of about ~3 dynes/cm. For the depletion of the leucocyte content of PRC, a CWST
greater than about 53 dynes/cm is desirable. The CWST may typically be above from about 53 dynes/cm to about 115 dynes/cm, but the invention should not be limited thereby. A more preferable CWST is above about 60 dynes/cm, and a still more preferable CWST
is from about 62 dynes/cm to less than about 90 dynes/cm.
If desired, the flow rate of biological fluid through the filter-can be regulated to obtain a total flow period of about 10 to 40 minutes by selecting the a~ iate element diameter, element thir~n~cs~ fiber diameter, and density, and/or by varying the diameter of the tube either u~ eam or downstream of the filter, or both up and downstream.
At these flow rates, l~llcocyte depletion efficiency in ~ s of 99.9% may be achieved. If P~P is the biological fluid being processed, these levels of efficiency may result in a PC product with less than about 0.1 x 106 leucocytes per unit of PC compared with the target of less than about 1 x 106.
The leucocyte depleting PRC ~OLOUS medium is primarily intended for use with PRC obtained from donated blood within about 8 hours of the time the blood was drawn. It may also be used to filter PRC
which has been stored at 4~C for up to several weeks, but, since the risk of clogging during filtration increases with storage age, the risk can be reduced, by for example, using pre-filters W092/076~ PCT/US91/08316 209~623 prece~ing the media described herein.
A PRC porous medium of the invention can be made to have a wide range of efficiencies for leucocyte depletion. If the porous medium is composed of 2.6~m fibers and weighs about P¦27-98 _ 29i200 V ~ grams where p = fiber density, grams/cc and V = ~oids volume, %, then when used for leucocyte depletion of PRC, the log of the efficiency, defined as the ratio of the influent l~u~G~Le ~Q~ LL~tion to the effluent leucocyte ~-o).~ L~ation, may be calculated from the formula lS Log efficiency = 25.S ~ V

In most applications, it is desirable to ~eep the time of flow of a unit of PRC through the porous medium when pressurized to about 30 to 300 mm of Hg to less than about 30 to 40 minutes; in order to achie~e this flow rate, the device should preferably be configured to a flow area of about 30 to 60 cm2.
For example, an 8.63 cm diameter (area =
58.5 cm2) ~ous medium made using 7.7 grams of 2.6~m diameter 1.38 g/c~ density fiber with a voids volume of 76.5%, would meet the requirements of equation (3), and its leucocyte depletion efficiency, in accordance with equation (4) would be log 6. Thus, if the influent concentration were 109 leucocytes/unit, then the effluent concentration would be ~ = 103 Similarly, if made with V = 88.2% using 2.6~m "'~92/076s6 PCT/US9t~08316 2(195623 diameter fibers of density 1.38 g/cc, the weight of the porous medium would be, per equation (3~:

1.38 127.98 - (29.26 x 180z ~]

= 3.0 grams, and the log of the efficiency would be, per equation (4~:
Log efficiency = 2S.5 ~l _ 880 = 3.0 Thus, if the influent leucocyte ~OI~ w ~ tion were 109 per unit of PRC, the effluent ~o~ Lation would be ~ = 106 per unit Equations (3) and (4~ are applicable to a voids volume range of a~out 73 to 88.5%, which spans the efficiency range from about log 3 to log 7.
Equations (3) and (4~ provide very useful guidelines for designing and building optimal or near to optimal PRC filters with limited experimentation; howeYer, a person familiar with the art will l~o~.,;ze that variations from these formulae and modifications to the porous media can be made to ~od~ce useful ~Gd~cts. Exemplary modifications and their effect on the performance characteristics of the ~o~ous media are set out ~elow:

, ~Og2~07656 pCT/US91/08316 2Q9~623 Desired Filter Changes from Characteristics ouations (3) and f41 Incr~ leucocyte 0 Reduce fiber diameter depletion efficiency 0 Increase weight of fi~er O Reduce voids volume Decrease probability of 0 Increase filter element 5 clogging area 0 Provide prefiltration 0 Incréase voids volume Decrease internal hold- 0 Decrease voids volume up volume O Eliminate prefiltration<
. 0 Use finer fiber(1~

Increase flow rate of 0 ~1 G~e5S the blood such the PRC that the PRC has lower hematocrit, hence lower viscosity 0 Use higher head when filtering 0 Increase filter area with concomitant reduction of thickness lS 0 Increase filter element voids volume Withstand higher O Reduce element voids applied differential volume pressure 0 Use coarser fiber (at the cost of reduced efficiency) 0 Use fiber with higher modulus W~92/076~ PCT/US91~08316-2~9S623 (1) Use of too small a fiber diameter may result in collArse of the filter element at normal working differential pressure.

(2) May result in excessively long filtration times, or complete clogging prior to completion of a transfusion.

RED C~TT BARRIER MEDIUN
Red cell barrier assemblies made in accordance with an emhcAiment of the invention and which are, for example, int~l~ose~ between the blood collection bag and the PRP bag, will generally remove about 85~
to 99% or more of the incident leucocytes, a removal rate that may not be sufficient to consistently achieve a residual leucocyte count of less than lO6 leucocytes per unit of PC. A principal function of thi8 assembly, however, is to act as an automatic ~valve~ during the decantation process by instantly stopping the flow of a biological fluid such as the ~u~eLIlatant layer (e.g., PRP), at the moment that red cells contact the porous medium comprising porous media. The mech~n;~m of this valve-like action is not well understood, but it may reflect ayyleyation of the red cells as they reach the porous surfâce, forming a barrier which prevents or blocks further flow of the supernatant layer through the porous medium.
AyyLeyation of red cells on contact with the porous filter ~rre~rS to be related to the CWST
and/or to other less well understood surface characteristics of the fibers which are generated by the herein described procedure for modifying the fibers. This theory for the proposed mech~nism is supported by the existence of filters capable of ~0~2/076~ PCT/US91/0~316 209~23 highly efficient leucocyte depletion of human red blood cell suspensions and which have pore sizes as small as 0.5~m, through which red cells pass freely and completely with no clogging, with applied pressure of the same magnitude as that used in the e~t invention.
On the other hand, the filters of the present invention, which typically have pore diameters larger than about 0.5~m, abruptly stop the flow of red blood cells when the ~ot~us medium is contacted by the red cells. This suggests that the valve-like action is not related to or CA~ by pore size or by a filtration mech~ni~m~- ~he mech~ni~m of this valve-like action is not well underctood, but it may reflect zeta potential-related ayy-~y~tion of the red cells as they reach the filter surface, forming a barrier which ~e~ s or blocks further flow of a biological ~1uid con~Ainin~ red cells through the porous medium.
In one emh~iment of the invention, the red cell filter assembly preferably includes a preferred range for the fiber surface area of about .04 to about .3 M2, and, more preferably, about .06 to about .20 M2. A preferred range for the porous medium flow area is about 3 to about 8 cmZ, and a more preferred range is about 4 to about 6 cm2. A preferred range for the voids volume is about 71~ to about 83S, and a more preferred range is from about 73% to about 80%. Because of its small size, a preferred device in accordance with this variation of the invention typically ~yh~hits a low hold-up volume. For example, when the biological fluid ~lo~essed is PRP, the device in accordance with this variation of the invention retains internally only about 0.5 to about 1 cc of PRP, ~ e_enting less than a 0.5% loss of platelets.

w092/076~ PCT/US9l/08316-. _ ~
209~523 In another variation of the devices of this in-vention, the PRP derived from a single unit of about 450 cc of human blood is passed, typically during a flow interval of about 10 to 40 minutes, through a filter comprising a ~vluUS medium, preferably comprising grafted fibers, with a surface area in the range of about O.OB to about 1.0 square meters, and more prefOEably about 0.1 to about 0.7 square meters, with a voids volume in the range of about lo 50% to about 89%, and more preferably about 60~ to about 85~. The filter element is preferably of right cylindrical form with the ratio of diameter to thic~ne~c preferably in the range of about 7:1 to about 40:1. The range of fiber diameter is prefer-red to be àbout 1.0 to about 4~m and is morepreferred to be in the range of about 2 to about 3~m. In relation to the previous variation of the invention, thi8 variation is made with higher fiber surface area, higher ~olvus medium flow area, smaller ~ ous medium density, and an increased voids volume.
All of these parameters can be varied; for example, the diameter of the ~vlOUS medium co~ be re~-lse~ and the thickness increased while re~ining 2S the same total quantity of fiber, or the fibers could be larger in diameter while increasing the total quantity of fiber, or the fibers could be p~ke~ as or~A,~A~ to preformed into a cylindrical disc. Such variations fall within the purview of this invention.
Another variation of this i..v~.Lion may comprise a ~OlG~S medium whèrein the u~Lleam portion is of a higher density than the downstream portion. For example, the porous medium may comprise a higher density upstream layer for blocking the passage of red blood cells and a lower 2 ~ 2 3 density downstream layer for the depletion of leucocytes. ~
In one embodiment of this invention, the fiber is surface modified in the same manner as in the pr~c~in~ versions, but the fiber surface area element is increased while, at the same time, the density is somewhat reduced. In this way, the automatic blockage of flow on contact by red cells -i~ combined with very high efficiency of leucocyte lo depletion.
A preferred range of fiber surface area for this variation of the invention is from about 0.3 to about 2.0M2, and a more preferred range is from about 0.35 to about 0.6N2. The upper limits of fiber surface area reflect the desire to accomplish the filtration in a relatively short time period, and may be increased if longer filtration times are acceptable. A preferred voids volume for the red cell barrier assembly is in the range of about 71~
to about 83%, and more preferably about 75~ to about 80~., A preferred flow area is from about 2:5 to about 10 cm2, and a more preferred area is from about 3 to about 6 cmZ. Leucocyte depletion efficiencies in ~ce~ of about 99.9~, which corresponds to an average residual leucocyte content per unit of less than about .05 x 1o6, can be obtained.
In a preferred embodiment of the invention, a ~o~ous medium for use with a biological fluid such as a supernatant layer (e.g., PRP) may compri~e the type of device disclosed in U.S. Patent 4,880,548.
In a preferred embodiment of the invention, a ~o~ous medium for use with a biological fluid such as a u8e sediment layer (e.g., PRC), may comprise the type of device disclosed in U.S. Patent 4,925,572 and U.S.
Patent 4,923,620.

W~s2~076~ PCT/US9l/08316-._ .
209~23 As noted above, as the sediment layer such as P~C is expressed from the collection bag, it may be ~oo~ eA through a device having a leucocyte depletion element in order to reduce the leucocyte content of the -~Aiment layer. In accordance with the invention, the ~o~o~s medium for removing leucocytes from the packed red cell component of a biological fluid comprises a leucocyte removal element or porous medium. The preferred element is typically made using radiation grafted melt blown fibers having an average diameter of from about 1 to about 4 ~m, preferably from about 2 to about 3~m.
Polybutylene terephthalate (PBT) web, which is a preferred material, may be hot comp~ to a voids lS volume of about 65% to about 90% and preferably to about 73% to about 88.5%.

SEPARATION A~ ~MRT.y The ~~ nt i,.v~.Lion involves the separation of one or more com~G~_~ts from a biological fluid.
In accordance with the ~--~nt i~v~lLion, a biological fluid, particularly blood, may be e~ A
to a separation medium suitable for passing at least one component of the biological fluid, particularly plasma, therethrough, but not other co~onents of the biological fluid, particularly platelets and/or red cells. Clogging of the separation medium by these other components is minimized or ~ ed.
In the emhcAiment of the il,v~ ion which includes a separation assembly 14, preferably a non-i~lLlifugal separation device, the ~u~e~llatant layer(e.g., PRP) may be r~ t~louyh a leucocyte depletion assembly, and then passed through the non-~ ifugal separation device 14, where it may ~e plo~ s~A and separated into components, which may be separately collected in container 15 and 20~5623 .

container 16. In a preferred embodiment, if the supernatant fluid is PRP, it may be separated into plasma and platelet concentrate as the PRP passes through the non-centrifugal separation device.
As shown in Figure 5, a preferred separation device of the present invention comprises a housing 210 having first and ~econd portions 210a, 210b joined in any convenient manner. For example, the first and seconA housing portions 210a, 210b may be joined by means of an adhesive, a solvent, or one or more connectors. The housing 210 also has an inlet 211 and first and second outlets 212 and 213, respectively, such that a first fluid flow path 214 is established between the inlet 211 and first outlet 212 and a ~e~on~ fluid flow path 215 is established between the inlet 211 and the second outlet 213. A separation medium 216 having first and second surfaces 216a, 216b is positioned inside the housing 210 between the first and ~econ~ housing portions 210a, 210b. Further, the separation medium 216 is positioned parallel to the first fluid flow path 214 and across the second fluid flow path 215.
Embodiments of the present invention may be configured in a variety of ways to ensure maximum contact of the biological fluid with the first surface 216a of ~?p~ration medium 216 and to reduce or eliminate clogging on the first surface 216a of the separation medium. For example, the separation device may include a first 5h~11 OW chamber facing the first surface 216a of thè separation medium 216.
The first chamber may include an arrangement of ribs which spread the flow of biological fluid over the - entire first surface 216a of the separation medium 216. Alternatively, the first chamber may include one or more channels, grooves, conduits, passages, or the like which may be serpentine, parallel, . , , .

2~623 ._ curved, or a variety of other configurations.
The fluid flow ç~nnels may be of any suitable design and construction. For example, the ~n~els may have a rectangular, triangular, or semi-circular cross section and a constant depth. Preferably, the çh~n~els have a rectangular cross ~ection and vary in depth, for example, between inlet 211 and outlet 212.
In the embodiment shown in Figures 6, 7, and 8, lo the inlet 211 of the housing 210 is connected to serpentine fluid flow c~n~els 220, 221, and 222 which face the first surface 216a of the separation medium 216. These rh~n~els 220-222 ~eparate the inlet flow of biological fluid into separate flow paths tangential to the first surface 216a of the separation medium 216. Extending along the first surface 216a, the serpentine fluid flow channels 220, 221, and 222 may be recombined at first outlet 212 of the housing 210.
Embodiments of the present invention may also be aonfigured in a variety of ways to minimize back pressure across the separation medium 216 and to ensure a ~ufficiently high velocity of flow to the second outlet 213 to ~event fouling of surface 216a, while minimizing hold-up volume. The separation device includes a second shallow chamber facing the second surface 216b of the separation medium 216. Like the fir~t chamber, the second chamber may include an arrangement of ribs or may comprise one or more ch~n~els, grooves, conduits, passages, or the like which may be serpentine, parallel, curved, or have a variety of other configurations.
The fluid flow channels may be of any suitable design and construction. For example, the ch~n~els may have a rectangular, semi-circular, or triangular W092/076~ PCT/US91/08316 -2U~5623 cross section and a constant or variable depth. In the embodiment shown in Figures 9~ everal serpentine fluid flow channels 231, 232, 233, 234, and 235 face the s~ronA ~urface 216b of the ~eparation medium 216. Ex*en~in~ along the cecond surface 216b, the serpentine fluid flow channels 231-235 may be recombined at the ~onA outlet 213.
Ribs, wall~, or projections 241, 242 may be used to define the rh~n~elc 220-222, 231-235 of the first and re~Q~ chambers and/or may ~y~L or position the separation medium 216 within the housing 210. In a preferred embodiment of the i..~,~ion, there are more wall~ 242 in the ~?con~
chamber than in the first chamber to ~Lev~lL
deformation of the separation medium 216 caused by pressure differential through the separation medium.
In use, a biological fluid, e.g., whole blood or-PRP, i8 fed under sufficient pressure into the inlet 211 of housing 210 from any ~uitable s~a of the biological fluid. For example, the biological fluid may be injected from a syringe into ~he inlet 211 or it may be forced into the inlet 211 from a flexible bag using a gravity head, a ~e_~ule cu~f, or an expressor. From the inlet 211, the biological fluid enters the ch~n~el~ 220-222 of the first chamber and pAsc~ tangentially or parallel to the first curface 216a of the separation medium 216 on the way to the first outlet 212 via the first fluid flow path 214. At least one com~G,.~-~ of the biological fluid, e.g., plasma, rACses through the separation medium 216, enters the ch-n~~lc 231-235 of the C~c~nA chamber, and is directed toward the second outlet 213 ~ia the second fluid flow path 215. As the biological fluid continues along the 3~ first flow path 214 tangentially or parallel to the first surface 216a of the separation medium 216, 20q~623 more and more plasma crosses the separation medium 216. A plasma-depleted fluid then exits the housing 210 at the first outlet 212 and is recovered in one container 217 while plasma exits the housing 210 at the second outlet 213 and is recovered in another container 218.
While any biological fluid cont~ini~g p may be used in conjunction with the present invention, the present invention is particularly well-cuited for use with blood and blood products, especially whole blood or PRP. By subjecting P~P to processing in accordance with the present invention, PC and platelet-free plasma may be obtained without centrifugation of the PRP and the attenA~t disadvantages discussed above. Likewise, platelet-free plasma may be obtained from whole blood. The biological fluid may be supplied in any suitable quantity consistent with the capacity of the overall device and by any suitable means, e.g., in a batch operation by, for example, a blood bag connected to an expressor or a syringe, or in a continuous operation as part of, for example, an apheresis system. Exemplary sources of biological fluid include a syringe 219, as shown in Figure S, or a biological fluid collection and processing system such as that disclosed in U.S. Patent 5,100,s6a, issued from application 07/609,654, filed November 6, 1990. A 80urce of biological fluid may also include an apheresi~ system, and/or may include a system in which biological fluid is recirculated through the system.
The separation medium and housing may be of any suitable material and configuration and the separation medium may be arranged in the present inventive device in any suitable manner so long as the biological fluid flow tangential or parallel to ., ~.Y

Wog2/07656 PCT/US91/08316 .
2~95623 the separation medium is maintained to a sufficient extent to avoid or minimize substantial platelet adhesion to the separation membrane. The llydr~yl,amics of flow parallel to a surface are indeed believed to be such that during flow parallel to the surface, platelets develop a spin which causes them to be recovered from the surface. While the preferred device has one inlet and two outlets, other configurations can be employed without adversely affecting the ~.v~e~ functioning of the device. For example, multiple inlets for a biological fluid may be used so long as the biological fluid flows tangentially across the face of the separation medium. The plasma may preferably be stored in a region separated from the separation medium in order to avoid possible le~eLse flow of the plasma back across the separation medium to the plasma-depleted fluid.
One ~ ed in the art will ~c~ e that platelet adhesion may be ~o.lL.olled or affected by manipulating any of a number of factors: velocity of the fluid flow, configuration of the c~nnel, depth of the c~nn~l~ varying the depth of the ch~h~l, the surface characteristics of the separation medium, the smoothness of the medium's surface, and/or the angle at which the fluid flow crosses the face of the separation medium, among other factors. For example, the velocity of the first fluid flow is prefera~ly sufficient to remove platelets from the surface of the separation medium.
Without in~n~;ng to be limited thereby, a velocity in ~ of about 30 cm/second has been shown to be adequate.
The velocity of the fluid flow may also be affected by the volume of the biological fluid, by varying the channel depth, and by the channel width.

~092/076~ PCT/US91/08316-2û~J~23 For example, the channel depth may be varied from about .25 inch to about .oOl inch, as shown in Figure ~. One skilled in the art will ~ ~J~i7e that a desired velocity may be achieved by manipulating these and other element~. Also, platelets may not adhere as r~ y to a separation medium having a smooth surface as compared to a membrane having a rougher surface.
In accordance with the il~v~.Lion, the separation medium comprises a ~OlO~S medium suitable for passing plasma therethrough. The separation medium, as used herein, may include but is not limited to polymeric fiber~ (including hollow fibers), polymeric fiber matrices, polymeric membranes, and solid porous media. Separation media according to the invention remove plasma from a biological solution con~n~n~ platelets, typically whole blood or PRP, without removing proteinaceous blood component~ and without allowing a substantial amount of platelets to pass theret~lv~h.
A separation medium, in accor~nc~ with the invention, preferably ~yh;hits an average pore rating generally or intrinsically smaller than the average size of platelets, and, preferably, platelets do not adhere to the surface of the separation medium, thus reducing pore blockage. The ~eparation medium should also have a low affinity for proteinaceous components in the biological fluid such as PRP. Thi~ ~nh~nces the l;kel ;hoo~ that the platelet ~o~ solution, e.g., platelet-free plasma will eXhibit a normal COl.~ -..LLation of proteinaceous clotting factors, growth factors, and other needed components.
For the separation of about one unit of whole blood, a typical separation device according to the invention may include an effective pore size smaller ~ ~'0g2~076~ - PCTtUS91/08316 .

209~623 than platelets on the average, typically less than about 4 micrometers, preferably less than about 2 micrometer~. The permeability and size of the separation device is preferably sufficient to produce about 160 cc to about 240 cc of plasma at reasonable ~.es~u~es (e.g., less than about 20 psi) in a reasonable amount of time (e.g., less than about one hour). In a~l~ance with the invention, all of these typical parameters may be varied to achieve a desired result, i.e., varied preferably to minimize platelet loss and to maximize platelet-free plasma production.
In accordance with the i,.v~ ion, a separation medium formed of fibers may be contin~ C, staple, or melt-blown. The fibers may be made from any material compatible with a biological fluid con~inin~ platelets, e.g., whole blood or PRP, and may be treated in a variety of ways to make the medium more effective. Also, the fibers may be bonded, fused, or otherwise fixed to one another, or they may simply be me~h~nically entwined. A
separation medium formed of a membrane, as the term is used herein, refers to one or more porous polymeric sheets, such as a ~o~l. or ,.oll uovell web of fibers, with or without a flexible porous suL~L~aLe, or may comprise a membrane formed from a polymer ~olution in a solvent by precipitation of a polymer when the polymer solution is contacted by a ~olvent in which the polymer is not soluble. The ~u-, polymeric sheet will typically have a substantially uniform, continuous matrix structure cont~ining a myriad of small largely inte~c~ .ected pores.
The separation medium of this invention may be formed, for example, from any synthetic polymer capable of forming fibers or a membrane. While not '"092/~76~ PCT/US91/08316-20~95623 n~c~s~ry to the apparatus or method of the invention, in a preferred embodiment the polymer is capable of serving as a substrate for grafting with ethylenically unsaLulated monomeric materials.
Preferably, the polymer should be capable of . reacting with at least one ethylenically unsaturated monomer under the influence of ionizing radiation or other activation means without the matrix being adversely affected. Suitable polymers for use as the substrate include, but are not limited to, polyolefins, polyesters, polyamides, polysulfones, polyarylene oxides and sulfides, and polymers and copolymers made from halogenated olefins and unsaturated nitriles. Preferred polymers are polyolefins, polyesters, and polyamides, e.g., polybutylene terephthalate (PBT) and nylon. In a preferred embodiment, a polymeric membrane may be formed from a fluorinated polymer such as polyvinyl;~r- difluoride (~v~). The most preferred separation media are a mi~lv~o~v~s polyamide membrane or a polycarbonate membrane.
Surface characteristics of a fiber or membrane can be affected as noted above for a ~osous medium.
An exemplary radiation grafting ~echn;que employs at least one of a variety of monomers each comprising an ethylene or acrylic moiety and a secon~ group, which can be selected from ~y~L~ ic ~ou~s (e.g., -COOH, or -OH) or h~d.~11obic ~ (e.g., a methyl group or saturated ~h~; ns such as -CE~CH~CH3~.
Grafting of the fiber or membrane surface may also be accomplished by com~o~,ds cont~n;~g an ethylenically unsaturated ~.v~, such as an acrylic - moiety, combined with a hydroxyl g~ù~, such as, h~dsoxyethyl methacrylate (HEMA~. ~se of HEMA as the monomer contributes to a very high CWST.
Analogues with similar characteristics may also be WO~2~076s6 PCT/US91/08316 2~93523 used to modify the surface characteristics of fibers.
In accordance with an emho~iment of the invention, the ~eparating medium may be surface-modified, typically by radiation grafting, in orderto achieve the desired performance characteristics, whereby platelets are ~ e,.L.&ted with a minimum of medium bloc~i~g, and whereby the resulting plasma solution contains essentially all of its native proteinaceous constituents. Exemplary membranes having a low affinity for pro~ein-ceous substA~c~
are disclosed in U.S. Patents 4,886,836; 4,906,374;
4,964,989; and 4,968,533, all in~ ~ted herein by reference.
I5 Suitable membranes in accordance with an emho~iment' of the invention may be microporous me~branes and may be ~ G~ced by a solution casting method.
As noted above, estab~ a tangential flow of the biological fluid h~q proceC-~~ parallel with or t~g~ntial to the face of the separation medium minimizes platelet collection within or p~ ge tl~ou~h the separation medium. In accordance with the i.,~nLion, the tangential flow can be induced by any me~h~n;cal configuration of the flow path which induces a high local fluid velocity at the immediate membrane surface. The ~ e_DuLe driving the biological fluid across the separation medium may be derived by any suitable means, for example, by gravity head or by an expressor.
The tangential flow of the biological fluid may be directed tangential or parallel to the face of the separation medium in any suitable manner, preferably utilizing a substantial portion of the separation medium surface while maint~ining a ~ W0~2J07656 PCT/US91/08316-20~S23 sufficient flow to ensure that the platele~s do not clog or block the pores of the separation-medium.
The flow of the biological fluid is preferably directed tangentially or parallel to the face of the separation medium through use of at least one ~el~e,.Line fluid flow ch~n~l which is designed to maximize utili7~tion of the separation medium, e..~e a sufficiently total area contact between the biological fluid and the separation medium, and maintain a sufficient flow of biological fluid to minimize or prevent platelet adhesion to the separation medium. Most preferably, several (e.g., three or more) fluid flow ch~n~Dl~ are U~ili7~ 80 as to fix the separation medium in place and to lS ~e,.L sagging of the membrane due to the applied pressure. The fluid flow channels may be of any suitable design and ~G..~Ll~ction and preferably are variable with e~2ct to depth such as depth to maintain optimal ~eO~le and fluid flow across the face of the separation medium. Fluid flow rh~
may also be utilized on the side of the separation medium opposite the biological fluid tangential flow to col.L ol the flow rate and ~e~ule drop of a platelet-poor fluid, such as plasma.
2S The ~ ent inventive device ~ay similarly be part of an apheresis system. The biological fluid to be ~ocec~ the platelet-rich solution, and/or the platelet-poor solution may be handled in either a batch or continuous manner. The sizes, nature, and configuration of the present inventive device can be adjusted to vary the capacity of the device to suit its intended environment.

GAS INIET/oUTLET
~nder certain circumstances, it may be desirable to maximize the recovery of a biological ~092/076s6 PCT/US9l/08316 .
209~623 fluid retained or entrapped in various elements of the biological fluid processing system. For example, under typical conditions, using a typical device, the biological fluid will drain through the system until the flow is s~orr~, leaving some of the fluid in the ~ystem. In one emhq~iment of the invention, the ret~ine~ fluid may be recovered by using at least one gas inlet and/or at least one gas outlet. An exemplary configuration of this embodiment is ~hown in Figure 3.
The gas outlet is a porous medium which allows gas that may be present in a biological fluid ~o~ ing sy~tem when the-biological fluid is ~o~ s~ in the system, out of the system. The gas 15 - inlet i5 a~porous medium which allows gas into a biological fluid processing system.
As used herein, gas refers to any gaseous fluid, such as air, sterilized air, oxygen, carbon dioxide, and the like; it is in~n~ that the invention is not to be limited to the type of gas used. .' The gas inlet and gas outlet are chosen so that the sterility of the system is not compromised. The gas inlet and the gas outlet are particularly suited for use in closed systems, or may be used later, for example, within about 24 hours of a system being opened.
The gas inlet and the gas outlet each comprise at least one porous medium designed to allow gas to pass therethrough. A variety of materials may be used, provided the requisite properties of the particular porous medium are achieved. These include the neceCcAry strength to h~l e the differential pressures encountered in use and the ability to provide the desired filtration c~r~hility while providing the desired permeability without the W092/0~6~ PCT/US91/08316-2n~5623 application of excessive pressure. In a sterile system, the ~ous medium should also preferably have a pore rating of about 0.2 micrometer or less to preclude bacteria r~C~ge.
S The gas inlet and gas outlet may comprise a porous medium, for example, a porous fibrous medium, such as a depth filter, or a porous membrane or sheet. Multilayered ~OLOUS media may be used, for example, a multilayered mi~o.~s membrane with one layer being liquophobic and the other rh; l ic.
Preferred starting materials are synthetic polymers including polyamides, polyesterc, polyolefins, particularly pol~ pylene and lS polymethylpentene, perfluorinated polyolefins, ~uch as polytetrafluoroethylene, polysulfones, polyvinylidene difluoride, polyacrylonitrile and the like, and compatible mi~L~ke_ of polymers. ~he most preferred polymer is polyvinylidene difluoride.
Within the al~s of polyamides, the preferred poly~erc include polyhexamethylene Adir~miae, poly-~-caprolactam, polymethylene sebacamide, poly-7-amino~ertanoamide, polytetramethylene adipamide (nylon 46), or polyhexamethylene azeleamide, with polyhexamethylene adipamide (nylon 66) being most preferred. Particularly preferred are skinless, substantially alcohol-insoluble, lly~l~hilic polyamide membranes, such as those described in U.S.
Patent 4,340,4~9.
Other starting materials may also be used to form the ~o~ous media of this invention including cellulosic derivatives, such as cellulose acetate, cellulose propionate, cellulose acetate-propionate, cellulose acetate-butyrate, and cellulose ~uLylate.
Non-resinous materials, such as glass fibers, may also be used.

W~92/076~ PCT/US91/08316 .

.

The rate of air flow through the gas outlet or the gas inlet can be tailored to the specific biological fluid or fluids of interest. The rate of air ~low varies directly with the area of-the porous medium and the applied pressure. Generally, the .area of the ~o~ous medium is designed to enable the biological fluid ~ r~cin~ system to be primed in a required time under the co~Aitions of use. For example, in medical applications it is desirable to be able to prime an intravenous set in from about 30 to about 60 ~ Ac, In such applications as well as in other medical applications, the typical ~o~ous medium is a membrane, which may be in the form of a disc ~c~ has a diameter from about 1 mm to about 100 mm, preferably from about 2 ~m to a~out 80 mm, and more preferably from about 3 mm to about 25 mm.
In accordance with the invention, the ~G- ~ cin~ system may be provided with a gas inlet to permit the i..~LV~uction of gas into the system, and/or with a gas outlet to permit gases in the various elements of the system to be separated from the biological fluid to be ~o~ . The gas inlet and the gas outlet may be used together in -ronn~ction with at least one assembly, porous medium, or container in the system, or they may be used separately.
To that end, a gas inlet or gas outlet may be included in any of the various elements of the biological fluid proc~ n~ system. By way of illu~Ll~Lion, a gas inlet or gas outlet may be included in at least one-of the CQ~ ;tS which connect the different containers, in a wall of the containers that receive the processed biological fluid, or in a port on or in one of those containers. The gas inlet or gas outlet may also be included on or in a combination of the elements W0~2/076~ PCT/US91/08316 2~95S2~

mentioned above. Also, an assembly or porous medium may include one or more gas inlet or gas outlet as described above. Generally, however, it i8 preferred to include a gas inlet or gas outlet in the conduits which connect the contAin~rs or in the functional medical device. Included within the scope of the invention is the use of more than one gas inlet or gas outlet in any ron~it, receiving con~ain~r, assembly, or porous medium.
It will be apparent to one skilled in the art that the placement of a gas inlet or a gas outlet may be optimized to achieve a desired result. For example, it may be desirable to locate the gas inlet ~D~eam of a porous medium and in or as close to lS the first container as is practical in order to maximize the ~eco~ery of biological fluid. Also, it may be desirable to locate the gas outlet downstream of the porous medium and as close to the receiving container as is possible in order to maximize the volume of gas that is removed from the system.
Such placement of the gas inlet or gas outlet is particularly desirable where there is only one gas inlet or gas outlet in the system.
In accordance with the i--~e..Lion, recovery from the various elements of the biological fluid processing system may be maximized. ~or example, whole blood is subjected to a ~lv.~r~Sin~ step, resulting in separate PRP and PRC layers. Then, the separate fractions of blood components are eA~ecsed to their respective receiving cont~in~rs t~ouyh the a~Lv~iate con~llits and ~o~ous media, if any.
Blood product that has become e..L~a~ed in these elements during processing may be recovered either by passing purge gas through the conduits and porous media, or by creating at least a partial vacuum in -the system to draw out the retained blood product , s3 and to permit it to drain into the appropriate receiving container or assembly.
The purge gas may be from any of a number of sources. ~or example, the biological fluid processing system may be provided with a storage container for the storage of the purge gas, the purge gas may be the gas that was removed from the system during the processing function, or the purge gas may be injected aseptically into the system from an outside source (e.g., through a syringe). For example, it may be desirable to use ~terile purge gas that has been sterilized in a separate container apart from the biological fluid processing system.
In accordance with the invention, a clamp, closure, or the like may be positioned on or in any or all of the conduits in order to facilitate a desired function, i.e., establ;~hing a desired flow path for biological fluid or gas. For example, when processing a biological fluid (e.g., PRP) through a system such as is illustrated in Figure 3, during the removal of gases from the conduits and the leucocyte depletion assembly, it may be desirable to clamp the conduit immediately below gas outlet 54.
When it is desirable to use the gas inlet 53 to maximize the recovery of a biological fluid, the clamp below gas outlet 54 is released, and a clamp in the conduit adjacent to gas intake 53 is opened.
As exempli~ied in Figure 3, other gas inlets and gas outlets (e.g., 51 and 52) may be operated in a similar manner.
With continued reference to ~igure 3, as a column of biological fluid (e.g., PRP) flows from the first container 11 through the conduit and the leucocyte depletion assembly 13, towards a satellite bag (not shown), it drives the gas in those elements towards the gas outlet 54.

~ , l ~ ~

W~92/076~6 PCT/US91/08316-.. , 2Q9~3623 The gas outlet may comprise a branching element with three legs. One leg may include a liquophobic porous medium which preferably has a pore size of not greater than 0.2 ~. At the br~n~in~ element, gas ahead of the column of biological fluid moves into one leg of the br~chi~g element. Since the gas r~s~s through the liquophobic ~o~ous medium, but the biological fluid does not, the gas ic separated from the PRP and is precluded from entering the satellite bag 15.
The gases separated by the gas outlet 54 may be vented from the system, or they may be collected in a gas container (not shown) and ~eL~,.ed to the ~ystem as a purge gas to facilitate the reoove~ of biological fluid that becomes trapped in the various components of the system.
After the system is primed and the gas outlet is inactivated, the clamp adjacent to the con~ino~s or assembly is or~n~A to allow the cont~ s to fill with ~looes~ biological fluid. This con~ c until the collection bag 11 coll~ps~. In order to recover the very valuable biological fluid ret~ine~ in the system, ambient air or a sterile gas may enter the system through gas inlet 51 or 53. If gas inlet 51 or 53 is a manual inlet means, a closure is opened or a clamp rele~eA; if the gas inlet 51 or 53 is automatic, the pressure differential between the gas inlet and the cont~in~rs will cause the air or gas to flow tl~uyl the cQ~ its~ through the p~ous media, and towards the respective containers. In the ~lo~e~s, retained biological fluid that is trapped in those elements during processing are recovered from those components and collected in the containers. It should be noted that the purge air or gas is preferably separated from the biological fluid at ~O9~/076~ PCT/US91/08316 2Q~23 5s gas outlet s2 or 54, so that little, if any, purge gas will be received ~y the con~in-~s~ This may be accompli~ y clamping the conduit downstream of the gas outlet 52 or 54. In another embodiment of the invention, the purge air or gas may be ~eparated from the system through a gas outlet located in the bag itself.

BRACKET
In another embodiment, the invention also includes a bracket which secures a filter assembly comprising a porous medium or one or more components of an assembly in place during ~.Llifugation so - that it (they) is (are) not damaged by the stresses generated during ~e.lLlifugation.
lS The blood collection and prore~ fiCi n~ assembly 10, With one or more satellite bags attached or cQ~n? ~ed via a ~on~it~ may be used integrally to separate components from who}e blood. This embodiment of the invention will be better understood by reference to the exemplary configuration _hown in Figure 4. During the centrifugation step in which the red cells are ron~e~trated at the bottom of the collection bag, forces of up to about 5000 times gravity (S000 G) or more may be generated. Therefore, the collection bag ic preferably flexible, as are the other bags, allowing them to settle to the bottom and against the walls of a centrifuge bucket 120, ~o that the bags th~celves are subject to little or no stress.
In contrast to the flexibility and pliability of the bags and tubing, the porous medium is typically contained in a rigid plastic housing (the combination of which is termed a filter assembly). The PRC housing is typically of larger 3S dimensions than the PRP housing, and is therefore W0~2/07656 PCT/US91/08316 , _ 2~9~623 s6 subject to an increased probability of suffering or causing damage during centrifugation. For example, a typical PRC filter assembly may weigh~about 20 grams (about .04 lbs), but its effective weight may be 5000 times greater, or about 200 lbs, under cellL.ifugation conditions of 5000 G. In conventional ~-nL~ifugation systems it is therefore difficult to avoid shattering the plastic housing.
Even careful placement of the PRC filter assembly in lo the ~enLlifuge bucket is likely to result in damage to the plastic tubing or to the bags. Furthermore, it is undesirable to enlarge the ~el.LLifuge h~cket to accommodate the filter assembly in the hv~t during the o~.L ifugation step, as this would not only require the use of a larger and more costly ~e..~Lifuge, but it would also require retrAin;n~ the th~C~s of blood processing ~ech~icians to e~e~ly assemble the blood bag sets into a new type of centrifuge bucket.
Accordingly, it is desirable that an im~o~e~
hl~o~ collection and processing system or set ~ho~ld be usable with existing centrifuge h~ck~ts. In accordance with the inv~ ion, this is preferably accomplished by locating the PRC filter assembly away from the greatest amount of G force; this is more-preferably o~tside or partly outside of the conventionally used ~ellL,ifuge hl~ket, in the manner shown in Figure 4.
In Figure 4 the bucket 120 depicts a centrifuge b~lcket such as is used in ~Ll~.L blood bank practice. These hllrkets are typically ~Ol~ ~L ~cted with heavy walls of high strength steel which enclose open space 121 into which the blood bag, its satellite bags, and the interposed tubing may be 3S placed. The bracket 122 used to hold a filter - assembly may be made of any high strength material, WOs2/076~ PCT/~S91/08316 203SS~3 s7 preferably metal or metallic alloy; titanium or stainless steel are more preferred for their ~-l~.yLh and the ease with which sanitary ron~itions can be maintAin~ The lower portion 123 of bracket S 122 is configured to ~ a~atively fit into cavity 121, preferably at a depth of about 0.5 to about 1 cm. Spring clips or other meanC may be used to position and/or retain bracket 122 in the bucket 120. ~lo~ve 124 located in the U~_l portion of bracket 122, is preferably configured to ~oo~e~atively accept the outlet port 125 of the filter assembly 114, and to allow the bottom portion of the filter assembly 114 to rest on the flat upper ~u,f~ces of bracket 122 adjacent to y~OOV~ 124. The is ~e~ l portion 126 of y oo~e 124 may be o~oLLioned such that port 125 of the filter assembly 114 fits into at least a portion of the ~-oo-~ 124 with a friction fit. The ends of y~oove 124 are preferably reduced to a width such that flexible tubing 112 ~ o;-e~ to the inlet and outlet of the filter assembly 114 i~ firmly retAin~, thereby helping to stabilize the filter assembly 114 when positioned onto bracket 122. The ~ Led portions of flexible t~lh~ng 112 then drop into the bucket in communication with the h~l~nce of the blood collection set contained therein. It is preferred that the bracket 122 retain the filter assembly 114 so that the plane of the ~o~ous medium is substantially ~e~ icular to 30''''~the G force created during operation of the ~.L,ifuge. Also, the bracket and filter assembly should be positioned on or in the cel~Lifuge bucket without interfering with the normal free-swinging action of the bucket 120 in the centrifuge during rotation.
Because the PRP filter is typically relatively W0~2/076~ PCT/US91/08316 _ 2 ~ 3 small and very light, it may be positioned within the bucket with the bags and the tubing. In another emho~iment of the invention, however, the groove 124 may be configured to hold more than one filter assembly, for example, both a PRC and a PRP filter assembly.
In anoth OE emh~i~ent of the invention, a larger bracket may be employed to hold a fir~t filter assembly and a ~?CO~ bracket holding a lo se~ filter asfiembly may be nested on top of the first bracket and filter assembly. One ckilled in the art will ~c-o~ e that various designs, configurations, and/or means may be employed to accomplish these f~.~ions.
Another feature of the invention is the location and manner in which the porous medium, particularly the PRC medium, is mounted on the c~.~ ifuge ~k~t during the ~,~Lifugation operation. Trials of a number of test filter-housings designed to fit within the ~el,~.ifuge h~k~t convi~cin~ly demo"~LLated that perforation of tubing lines by the housing is a frequent G~uLlence during centrifugation. Also, it is very difficult to design a housing that can reliably withstand d without shattering. Further, the existing centrifuge buckets are designed to carry the c~v~ional blood collection sets, which i.,~ol~.ate no filter elements. Fitting the added bulk of a PRC filter assembly into a conventional bucket was, thus, very difficult. These very serious problems were eliminated ~y mounting the PRC
filter assembly on a bracket outside of the bucket.
This provides adequate support for the filter assembly by cooperatively arranging flange portion 127 of bracket 122 (Figure 4) to accommodate the contours of the centrifuge bucket. Furthermore, -bracket 122 is preferably positioned above the top of the ~ucket, a location which is much closer to the center of rotation of the centrifuge that the force to which the filter assembly is subjected is about 40% to about 60% that of the bottom of the bucket 120. Additionally, the narrow slots at each end of the brac~et hold the tubing connections firmly, and permit the tubes to drop back into the bowl. Su~lisingly, the suspended portions of the tubing tolerate the centrifuging operation very well.
A system according to the present invention may be used in conjunction with other assemblies or porous media, including filtration and/or separation devices, e.g., a device for removing leucocytes from a platelet-cont~ g solution or concentrate.
Exemplary devices are disclosed in U.S. Patent 4,880,548, and U.S. Patent 4,925,572.
Housings can be fabricated from any suitably impervious material, including an impervious thermoplastic material. For example, the housing may preferably be fabricated by injection molding from a transparent or translucent polymer, such as an acrylic, poly~L~lene, or polycarhon~e resin.
Not only is such a housing easily and economically fabricated, but it also allows observation of the passage of the fluid through the housing.
The housing into which the ~ous medium is ~ealed or interference fit is designed to achieve convenience of use, rapid priming, and efficient air clearance.
While the housing may be fashioned in a variety of configurations, the housing of a porous medium according to the present invention preferably comprises a housing such as that disclosed in U.S.

..

W0~2~076~ PCT/US91/08316 2ass62~

Patents 4,880,548; 4,923,620; and 4,925,572, which are generally similar in configuration to housing 114 in Figure 4.
A number of additional contAin~s may be in communication with the biological fluid processing ~ystem, and can be utilized to define different flow paths. For example, an additional satellite bag contAini~q physiological solution may be placed in communication with the biological fluid processing system u~ eam of the leucocyte depletion assembly (e.g., through the gas inlet), and the solution may be pACse~ thlG~h the leucocyte depletion assembly so that the biological fluid that was held up in the assembly can be collected.
Similarly, a satellite bag contAining physiological solution may be placed in communication with the biological fluid processing system downstream of the leucocy-te depletion assembly (e.g., tl~ou~l. the gas outlet), and the ~olution may be rA~ tl~vuyh the leucocyte depletion assembly 80 that the biological fluid that was held up in the assembly can be later collected.
It will be appreciated that when the biological fluid from the collection bag 11 is e~lessed towards the containers, some of the biological fluid may be trA~r~ in the conduits and/or the ~vLOUS
mediums. For example, 8cc to 35cc is typically retAi~e~ in the system; but as little as 2cc to as much as 150cc or more may be retained in some ~y~eo of systems.
In an emhc~iment of the invention (not shown), air or gas may be stored in at least one gas cont~in~r; upon opening of valve or clamp means in the conduits, gas can be fed through them to purge 3s the conduits and assemblies, thereby facilitating the recovery of biological fluid that may have been ~ W0'92~076~ PCT/US9l/08316 .

209~623 trapped during processing.
Preferably, the purge air or gas is fed to the conduits at a point as close as is reasonably possible to container 11 to maximize the volume of biological fluid recovered. The air or gas container is preferably flexible so that the gas therein may be fed to the system merely by simple compression. The biological fluid containers and the air or gas containers may be composed of the same material.
Priming, as used herein, refers to wetting or priming the inner surfaces of a device or assembly prior to its actual use allowing a separate assembly to be injected into the ~y~L~m. A valve or clamp is opened to allow fluid to flow t~lou~ll the assembly;
then, with the r~cc~ge of fluid through the assembly, gas downstream of the fluid is expelled ~ough the gas outlet until fluid reaches a ~L~ g element, at which point the clamp is closed. With the clamp in a closed position, the ro~e~tor downstream of the gas outlet may be opened or readied for use without fluid in the assembly dripping through the connector.
In accordance with the invention, the biological fluid collection and processing assembly should be able to withstand rigorous sterilization and ce~.~ ifugation environments, typically consisting of radiation sterilization (at about 2.5 megarads), and/or autoclaving (at about 110~C to about 120~C for about 15 to 60 minutes), and/or centrifugation (typically about 2500 to 3500 G for about 5 to 15 minutes; however, depending on which biological fluid component is intended to have maxim~m recovery, the centrifugation may be about 3S 5000 G for about lo to 20 minutes).

209~ 3 The invention also involves a method for collecting and processing blood comprising collecting whole blood in a contAin~; centrifuging the whole blood; passing the supernatant layer of the centrifuged blood through a first porous medium, the first porous medium comprising at least one of a leu_o~e depletion medium, a red cell barrier medium, and a combined leucocyte depletion red cell barrier medium; and passing the ~e~iment layer of the ~e"L~ifuged blood through a ~erQn~ porous -medium, the second porous medium comprising a leucocyte depletion medium.
m is invention may also include a method for ~ e~ing a biological fluid comprising passing a biological fluid from a first container to a first porous medium comprising a red cell barrier medium, wherein the biological fluid r~s~ in a first flow path; and passing a biological fluid from a first container to a ~econ~ ous medium comprising a - 20 leucocyte depletion medium, wherein the biological fluid passes in a second flow path.
In general, donated whole blood is processed as soon as practicable in order to more effectively reduce or eliminate contaminating factors, including but not limited to leucocytes and microayy.eyates.
Leucocyte depletion has been heretofore typically performed during transfusion at bedside, however, in accor~nc~ with the subject in~ention leucocyte depletion is accomplished during the initial processing of the whole blood, which in United States practice is generally within 8 hours of collection from the donor. After the red cells have been sedimented by centrifuging, the supernatant PRP is expressed from the blood collection bag into a first satellite bag through one or more porous media which diminish the amount '.W092/076~ PCT/US91/08316 .
20~5~23 of leucocytes and/or block red cells, and the P~c remaining in the blood collection bag is then passed through a porous medium which removes leucocytes into a fiecon~ satellite bag.
In general, using the Figures for reference, the biological fluid (e.g., donor's whole blood) is received directly into the collection bag ll. The collection bag ll, with or without the other elements of the system, may then be c~ ifuged in order to separate the biological fluid into a ~u~_ ~.atant layer 31 and a ~-~;ment layer 32. After ~n~ifugation, if whole blood is used, the ~ e~..atant layer is prima~ily PRP, and the ~-~;ment layer is primarily PRC. The biological fluid may be e~ o~e~ from the collection bag as separate -~u~e~..atant and ~e~i~cnt layers, respectively.
There may be a clamp or the like between the collection bag ll and the flexible tubing 25, or within the tubing, to ~vel-~ the flow of the ~u~Lllatant layer from entering the wrong conduit.
Movement of the biological fluid thro~gh the system is effected by maint~ining a pressure differential between the collection bag and the destination of the biological fluid (e.g., a cont~ine~ such as a sa~ te bag or a needle on the end of a conduit). The system of the invention is ~uitable for use with ~ol,ventional devices for establi~hing the pressure differential, e.g., an e~ or. Exemplary means of es~hl;~ing this pressure differential may be ~y gravity head, applying pressure to the collection bag (e.g., by hand or with a pressure cuff), or by placing the other container (e.g., satellite bag) in a chamber (e.g., a vacuum chamber~-which establishes a pressure differential ~etween the collection bag and the other container. Also included within the scope ~92/076~ PCT/US91/08316 2~S623 of the invention may be expressors which generate substantially equal pressure over the entire collection bag.
As the biological fluid passes from one bag to S the next, it may pass tl~ou~h at least one ~OLOUS
medium. Typically, if the biological fluid is the supernatant layer (e.g., P~P), it may pass from the collection bag through one or more devices or assemblies comprising one or more porous media -- a leuco~e-depletion medium, a red cell barrier-medium, a porous medium which combines the red cell barrier with leucocyte depletion in one porous medium, or a leucocyte depIetion medium and a red cell barrier medium in series. The supernatant layer 31 is ~ e_~ed from the first container 11 until flow is stopped, typically by closing a clamp in conduit 20, or automatically if the assembly includes a red cell barrier medium 12. Preferably, the ~e~,atant layer pA-C~- through a red cell barrier medium and then t~ h a leucocyte depletion medium. The supernatant layer is then leucocyte-depleted after passing through the leucocyte depletion medium. Additional processing, if desired, may occur downstream of the leucocyte depletion medium, either connected to the system or after being separated from the system.
The sediment layer 32 in collection bag 11 may be r~-cee~ through a leucocyte depletion assembly 17 and into a container 18, such as a satellite bag.
Typically, the collection bag 11, now contA~n;ng primarily red cells, is then subjected to a pressure differential, as noted above, in order to prime the leucocyte depletion assembly 17 and to initiate flow.
In accordance with an additional embodiment of the invention, a method is provided whereby the W~s2/076~ PCT/US9l/08316 .

~0~623 6s recovery of various biological fluids trapped or retained in various elements of the system is maximized, either by causing a volume of gas heh;n~
the ~a~ed or ret~in-~ biological fluid to push the fluid through those elements and into the designated con~ , assembly, or porous medium, or by drawing the ~ed or retained fluid into the designated con~ r, assembly, or porous medium by pressure differential (e.g., gravity head, pressure cuff, suction, and the like~. Thifi provides for a more complete emptying of the con~in~ assembly, or ~OI~ medium. Once the container is emptied completely, the flow is stopped automatically.
In order that the invention herein described may be more fully understood, the following examples are set out regarding use of the present invention.
These examples are for illustrative ~u~o~es only and are not to be construed as limiting the present invention in any manner.

, Examples Exam~le 1 The biological fluid proce~cing system used to perform the first example includes a blood collection bag, separate PRP and PRC leucocyte depletion ~CSD -hlies, as well as separate PRP and PRC satellite bags. In addition, a red cell barrier medium between the collection bag and the PRP
leucocyte depletion assembly precludes the flow of red cells into the PRP satellite ~ag.
The red cell barrier assembly includes a ~OlOUS
medium for blocking flow upon contact by red cells when the PRP passes from the collecting bag. The red cell barrier assembly was preformed from 2.6~m average diameter PBT fibers, which had been surface modified in accordance with the procedures disclosed ~ 92/076~6 PCT/US91/08316 ~
209~62t, in U.S. Patent 4,880,548, using hydroxyethyl methacrylate and methacrylic acid in a monomer ratio of .35:1 to obtain a CWST of 95 dynes/cm and a zeta potential of -11.4 millivolts. The ~vLOUS element's effective diameter was 2.31 cm, presenting a filter area of 4.2 cm2, thickness was .051 cm, voids volume was 75~ (density, 0.34 g/cc), and fiber surface area was .08m2.
The volume of PRP held up within the housing was <0.4 cc, representing a loss of PRP due to hold-up of less than 0.2%. The flow stopped abruptly as red cells reached the u~ eam surface of the red cell barrier assembly, and there was no visible evidence of red cells or hemoglobin downstream.
The PRP leucocyte depletion assembly cont~ g a ~o~ous medium for dépleting leucocytes from PRP
after the PRP r~sc~ through the red cell barrier assembly, is described in U.S. Patent 4,880,548.
Similarly, the PRC leucocyte depletion assembly, con~ in~ a porous medium for depleting leucocytesfrom the unit of PRC is described in ~.S. Patent 4,925,572. The Example used a single unit of whole blood donated by a volunteer. The unit of blood was collected in a collection bag which was pre-filled with 63 ml of CPDA anti-coagulant. The collected blood was subjected to soft-spin centrifugation in accordance with customary blood bank practices. The collection bag was transferred, with care to avoid di~L~l~ing its contents, to a plasma extractor, which was spring biased to develop a pressure of about 9o mm Hg.
The pressure from the expressor drives the PRP
from the collection bag through the red cell barrier assembly, the PRP filter assembly ~where it is leucocyte depleted), and then to the satellite bag.
As the P~P exited the collection bag, the interface W0~2/07656 PCT/US91/08316 209~523 6~
between the PRC and PRP rose. When the red cells (present in the leading edge of the PRC layer), contacted the red cell barrier assembly, the flow was terminated, automatically, and without monitoring.
The PRC remaining in the collection bag is also sed. The collection bag is susr~Ae~ and then squeezed to prime the PRC leucocyte depletion filter, and the P~C is leucocyte depleted. When the ~uspended collection bag is empty, the ~G~SS stops automatically. The now leucocyte depleted red cell ~lcduct is eventually collected in the satellite bag, and is available for transfusion into a patient as required.
The PRP previously collected in the satellite bag was then processed using normal blood bank procedures (i.e., hard-spin centrifugation) to ~Gd~ce PC and plasma.

Exam~le 2 Whole blood was collected into an Adsol~ donor set and was processed under s~n~Ard conditions to yield a unit of PRP. The PRP was then filtered to remove leucocytes using a filter device described in U.S. Patent 4,880,548. The removal efficiency was ~99.9%.
The filtered PRP unit was then placed in a pressure cuff to which a pressure of 300 mm Hg was applied. The tubing exiting the bag (clamped closed -at this point) was connected to the inlet port of a separation device as shown in ~igures 5, 6, and 9.
A microporous polyamide membrane having a pore rating of 0.65 microns was used as the separation medium in the device. The area of the membrane was about 17.4 square centimeters. The depth of the first fluid flow path channels decreased from about ~ 92/076~6 PCT/US9l/08316 2~9~623 0.03 cm near the inlet to about O.ol cm near the outlet. The depth of the second f luid-flow path ~h~nnel~ was-about 0.025 cm. The outlet ports of the device were connected to tubing which allowed the volume of f luid exiting the device to be measured and saved for analysis.
The test of the present invention was started by ~r~ning the clamp and allowing PRP to enter the device. Clear fluid (plasma) was observed to exit one port, and turbid f luid (platelet concentrate~
exited the other port. The duration of the test was 42 mLnutes, during which 154 ml of plasma and 32 ml of platelet concentrate was collected. The ~u, ~n~ ~ion of platelets in the plasma was found to be 1.2 ~ 1041~1, while the concentration of platelets in the platelet concentration was found to be 1.43 x 106/~l.
The above results indicate that platelets can be c~nc~.L,ated to a useful level and plasma can be recovered in a reasonable time by a device according to the invention.

Example 3 Whole blood was collected and processed into blood components as in example 2, and compared to 2S that produced by conventional processing. The results comparing the blood component volumes to their respective leucocyte counts are listed in Table I, which shows the increased efficiency of leucocyte removal over the conventional procedures.
Table I also reflects the increased yield of plasma and corresponding decreased yield of PRC resulting from the use of this invention.

~O9~n76~ PCT/US91/08316 ' ~ 2~95623 ~AsLE I
Conventional Invention Whole Blood volume (cc) 450 - 500 450 - 500 WBC-whole blood 2 x 109 2 X 109 PC volume (cc) 50 - 65 50 - 65 WBC-PC ~1 x 108 zl x 105 Plasma volume ~cc) 165 - 215 170 - 220 WBC-Plasma <lot <lo8 PRC Volume (cc~* 335 320 WBC-PRC* 2 x 109 1 x 106 * w/AdsolTM
, Exam~les 4-8 -The blood collection sets used to perform the examples were in general conformance with Figure 1, without the optional red cell barrier medium assembly, and the procedure was as descri~ed earlier, using an apparatus in accordance with Figure 4 for the first centrifuging step.
The porous medium for depleting leucocytes from PRP was preformed into a cylindrical filter element 2.5 cm in diameter and 0.33 cm thick, using PBT
fibers 2.6~m in average diameter and of density l.38 g/cc, which had been surface modified in accordance with the procedures disclosed in U.S. Patent 4,880,S48, using a mixture of hydroxyethyl methacrylate and methacrylic acid monomers in a weight ratio of 0.3:l. The porous medium had a CWST
of 95 dynes/cm and a zeta potential of -ll.4 millivolts at the pH of plasma (7.3~. Fiber surface area was 0.52 M2 and voids volume was 80%.
The porous medium for depleting leucocytes from ~92/07656 PCT/US91~08316 ._ .
209~623 PRC, in accordance with equations (3) and (4) above, was calculated to obtain a leucocyte depletion - efficiency better than log 3 (i.e. >99.9% reduction of leucocyte content). This was accomplished by using 3.4 grams of 2.6~m diameter PBT fiber, about 13~ more fiber than called for by eguations (3) and (4), and, in order to further increase the leucocyte depletion efficiency, the voids volume was decreased to 81%. These changes boosted the efficiency to better than log 4 (i.e., to >99.99%). When the PRC
was expressed through this filter medium contained in a housing 6.4 cm in diameter, flow times in the desired 10 to 40 minute range were obt~inc~ at go mm Hg applied ~t e_DU~e- The fiber surfaces had been modified in the manner disclosed in U.S. Patent 4,925,572, using a mixLule of l.yd~oxyethyl methacrylate and methyl methacrylate in a weight ratio of 0.27 to 1; the porous medium had a CWST of 66 dynes/cm.
me PRC ~vLO~S medium described above was -preceded by a pre-filter consisting of five layers of PBT melt blown web laid up in the order noted below to a total thickness of 0.25 cm:

w092io76~ PCT/US91/083a6 .
2û9S623 - Fiber Weight, Diameter, Grade mq~cm2 ~m CWST

2.0-0.6 .002 12 So 2.0-l.o .002 9 50 2.5-3.5 .003 4.5 66 5.6-7.1 .006 3.0 66 5.2-10.3 .006 2.6 66 Each of the examples used a single unit of lo blood donated by a volunteer. The unit of blood was collected in a blood collection set configured as shown in Figure 1 (without the optional red cell barrier medium assem~bly), the collection bag of the set having been pre-filled with 63 ml of CPDA anti-coagulant. The volume of blood collected from thevarious donors is shown in Column 1 of Table II.
The collection set was positioned into the ~el.L.ifuge bucket of Figure 4 in accordance with customary blood bank practice, except that the PRC
filter was assembled to the bracket, which in turn was mounted on the upper portion of the centrifuge h~ et, thus ~ecuring the PRC filter outside and above the centrifuge bucket.
~he centrifuge was then rotated at a speed that developed 2280G (at the bottom of the bucket) for 3 minutes, sufficient to cause the red blood cells to ~iment into the lower portion of the collection bag. The bracket was then remo~ed and the collection bag was transferred, with care to avoid disturbing its contents, to a plasma extractor, which was spring biased to develop a pressure of about so mm Hg. Breaking the seal connecting the collection bag to the PRP filter and then breaking the seal connecting the PRP filter to the satellite ~ 92/076~ PCT/US91~08316 21)95623 ~ag permitted the PRP supernatant layer to flow from the collection bag through the PRP filter into the satellite bag. As the PRP exited, the interface between the PRC and PRP rose in the collection bag, with flow continuing for about lO to 20 minutes until all of the PRP had rA~c~A through the PRP
filter, at which time the flow terminated abruptly and automatically as the leading edge of the PRC
layer reached the PRP filter. The tubing was then clamped adjacent to the collection bag, and adjacent to the satellite bag, and the tubing between the two clamps and the PRP filter was cut. The PRP
- collected in the satellite~bag was then proce~s~ -using normal blood bank ~ vcedures to ~IGd~ce leucocyte-depleted PC and plasma. The volumes of PC
and plasma are shown in Table II along with the number of residual leucocytes in the PC.
The collection bag, now contAining only the sedimented red cells, was removed from the plasma extractor, and lO0 ml of AS3 nutrient solution, which had been preplaced in the other satellite bag, was transferred into the collection bag through the PRC filter. The contents of the collection bag were then thoroughly mixed. With about 120mm Hg pressure applied by gravity head, the PRC in the collection bag was next leucocyte-depleted by passing it through the PRC filter to the satellite bag. The PRC was now available for transfusion into a patient as required. The volume, hematocrits, and the residual leucocyte counts in the PRC are listed in Table II.
The leucocyte counts presented in the table reflect the sensitivity of the methods used for assaying the number of leucocytes residual in the PRC and in the PC effluents. No leucocytes were in fact detected in the leucocyte depleted effluents of .W0~2/07656 PCT/US91/08316 - 209~iS2~

any of the examples. Parallel experiments using more sensitive (but more laborious) assay methods indicate that the leucocyte depletion efficiencies were about ten to one hundred time ~etter than is indicated by the data presented in the table.

TABLE ~
Test 1 Test 2 Test 3 Test 4 Test S
Whole Blood CQ11~ (mL) 407 387 399 410 410 lo Whole Blood Hct (%) 45 42 5 40 41 38.5 PRP Filtration Time (min.) 16 11 14 15 19 PRP Volume (mL) 211 173 196 177 232 PC Volume (mL) 47 52 49 69 61 R~ d~
WBC-PC*<l.Oxl05cl.lXlo5<l.lx105 <1.5Xlo5 <1.;3xlo5 20 PRC Filtration Time (min.) 1S 18 11 11 12 PRC Volume (mL) 28s 318 301 306 288 PRC Hct (%)64.5 67 S1 52.5 60.5 Residual WBC-PRC~ c7.3x1~ <8.0x106 <7.5x1~ <7.7xl~ <7.2x106 -- * per unit ~92/07656 PCT/US91/08316 2095~23 While the invention has been described in some detail by way of illustration and example, it should be understood that the invention is ~n~c~rtible to various modifications and alternative forms, and is S not Ie~icted to the specific embodiments set forth . in the Examples. It should be understood that these specific embodiments are not intended to limit the invention but, on the ~oll~ary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (42)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A biological fluid processing system comprising: a first container; a first porous medium comprising a red cell barrier medium communicating with the first container; and a separation medium downstream of and communicating with the first porous medium.

2. A biological fluid processing system comprising: a first container; a first porous medium communicating with the first container, the first porous medium comprising one or more of a leucocyte depletion medium, a red cell barrier medium, and a combined leucocyte depletion red cell barrier medium; and a second porous medium communicating with the first container, the second porous medium comprising a leucocyte depletion medium.

3. The system of claim 2, wherein the first porous medium and the second porous medium comprise fibers that have been modified by exposure to a monomer comprising a polymerizable group and a hydroxyl-containing group.

4. The system of claim 3, wherein the fibers of the first porous medium have been modified with a mixture of monomers comprising hydroxyethyl methacrylate and methacrylic acid.

5. The system of claim 4, wherein the methacrylic acid to hydroxyethyl methacrylate monomer weight ratio in the modifying mixture is between 0.01:1 and 0.5:1.

6. The system of any one of claims 1 to 5, wherein the first porous medium is structured to permit platelets to pass therethrough but is structured to block red blood cells from passing therethrough.

7. The system of any one of claims 1 to 6, wherein the first porous medium comprises a fibrous medium and wherein the fiber surface area of the first porous medium is 0.3 to 2.0 M2.

8. The system of any one of claims 1 to 6, wherein the first porous medium comprises fibers and the fiber surface area of the first porous medium is 0.35 to 0.6 M2.

9. The system of any one of claims 1 to 6, wherein the first porous medium comprises fibers and the fiber surface area of the first medium is 0.04 to 0.3 M2.

10. The system of any one of claims 1 to 6, wherein the first porous medium comprises a fibrous medium and wherein the fiber surface area of the first porous medium is from 0.08 to 1.0 M2.

11. The system of any one of claims 1 to 10, wherein flow area of the first porous medium is 2.5 to 10 CM2.

12. The system of any one of claims 1 to 11, wherein the first porous medium has a voids volume of 71% to 83%.

13. The system of any one of claims 1 to 11, wherein the first porous medium has a voids volume of 50% to 89%.

14. The system of any one of claims 1 to 11, wherein the first porous medium has a voids volume of 60% to 90%.

15. The system of any one of claims 1 to 14, wherein the first porous medium has a zeta potential of from -3 to -30 millivolts at a pH of 7.3.

16. The system of any one of claims 1 to 15, wherein the first porous medium has a CWST of greater than 70 dynes/cm.

17. The system of any one of claims 2 to 16, wherein the second porous medium has a CWST of greater than 53 dynes/cm.

18. The system of any one of claims 2 to 17 wherein the second porous medium also includes a prefilter.

19. The system of any one of claims 1 to 18, further comprising one or more gas inlets and one or more gas outlets.

20. The system of any one of claims 2 to 19, wherein the first and second porous media comprise synthetic polymeric fibers.

21. The system of claim 20, wherein the first and second porous media comprise polybutylene terephthalate fibers.

22. The system of any one of claims 1 to 21, wherein said system is a sterile, closed system.

23. The system of any one of claims 1 to 22, wherein the system is structured to permit processing biological fluid within about 8 hours.

24. The system of any one of claims 1 to 23, including one or more pre-filters upstream of the first porous medium.

25. The biological fluid processing system of any one of claims 1 to 24, including a red cell barrier medium structured to permit separation of a red cell-containing portion of the biological fluid from a non red cell-containing portion of the biological fluid.

26. A biological fluid processing system for use with a centrifuge comprising: one or more containers which fit in a bucket; one or more filter assemblies in fluid communication with the one or more containers; and a bracket adapted to receive the one or more filter assemblies and structured to be positioned on or in the bucket.

27. A method for processing biological fluid comprising: separating the biological fluid into a supernatant layer and a sediment layer; passing the supernatant layer of the biological fluid through a first porous medium, the first porous medium comprising one or more of a leucocyte depletion medium, a red cell barrier medium, and a combined leucocyte depletion red cell barrier medium;
and passing the sediment layer of the biological fluid through a second porous medium, the second porous medium comprising a leucocyte depletion medium.

28. The method of claim 27, further comprising expressing the supernatant layer to the first porous medium and expressing the sediment layer to the second porous medium concurrently or sequentially.

29. The method of claim 27, further comprising passing the supernatant layer through a separation assembly.

30. The method of claim 27, 28 or 29, further comprising introducing gas through a gas inlet and expelling gas through a gas outlet.

31. The method of any one of claims 27 to 30, wherein passing the biological fluid through a first porous medium comprises passing the supernatant layer through a red cell barrier medium or a combined leucocyte depletion red cell barrier medium until the first porous medium is blocked.

32. The method of any one of claims 27 to 31, wherein the biological fluid is processed through a sterile closed system.

33. The method of any one of claims 27 to 32, in which the biological fluid is processed within about 8 hours.

34. The method of any one of claims 27 to 33, wherein the supernatant layer of the biological fluid is passed through one or more pre-filters prior to passing the supernatant layer of biological fluid through the first porous medium.

35. A method for collecting and processing a biological fluid comprising: collecting a biological fluid in a first container of a biological fluid processing system comprising a first container, a second container, and a filter assembly interposed between the first container and the second container; placing the first container holding whole blood and the second container in a centrifuge bucket; positioning the filter assembly away from the bottom of the centrifuge bucket; and centrifuging the system.

36. The method of claim 35, wherein positioning the filter assembly includes positioning the filter assembly on a bracket which rests on or partly within the centrifuge bucket.

37. The method of claim 35 or 36, in which the biological fluid is processed within about 8 hours.

38. A method for processing a biological fluid comprising: separating the biological fluid into a supernatant layer and a sediment layer; passing the supernatant layer of the biological fluid through a first porous medium comprising a red cell barrier medium until the red cell barrier is blocked; and then, passing the supernatant layer through a separation medium.

39. A method for processing a biological fluid comprising: separating a biological fluid into a supernatant layer and a sediment layer in a first container; separating the supernatant layer from the sediment layer by passing the supernatant layer from the first container through a porous medium to a second container; and, passing the sediment layer toward a third container.

40. A method of producing a platelet suspension substantially free of red cells comprising: separating a biological fluid into a supernatant platelet-containing suspension and a sediment red cell containing suspension in a first container; separating the supernatant suspension from the sediment suspension by passing the supernatant from the first container through a porous medium to a second container; and, passing the sediment suspension toward a third container.

41. The method of claim 40, further comprising separating the supernatant suspension from the sediment suspension by passing the supernatant suspension through the porous medium until the porous medium is blocked.

42. The method of claim 40 or 41 further comprising recovering a platelet suspension substantially free of red cells.