CA2086516C - Methods for processing red blood cell products for long term storage free of microorganisms - Google Patents

Abstract

A blood processing method provides a red blood cell product that is free of microorganisms like Yersinia enterocoli-tica during storage periods over 24 hours. The red blood cell product is collected in a first container (16), where it is refrige-rating to cool the blood product to a temperature of about 3 to degrees C. The refrigerated product is transferred from the first container (16) into a storage container (34) through a pre-scribed filter medium (44) that comprises a mass of synthetic fibers having an average fiber diameter of about 10 microns or less and a bulk density of about 0.7 gram per cubic centimeter or less. The filtered product is retained in the storage container (34) at a temperature of about 3 to 5 degrees C for a storage pe-riod over 24 hours after filtration. Using the filter medium (44), microorganisms like Yersinia enterocolitica present in the red blood cell product at the time of collection are depleted. The filtered blood product remains free of clinically significant numbers of microorganisms throughout refrigerated storage up to time of transfusion:

Description

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The 3aav~ntioa~ g~n~~°~lly xel~t~s ts~ blood coll~ct~.on and p~oo~~~irag methods. Try ~ n~or~y~rticu~
lay s~r~s~, the invention ~~l~t~~ to ~erthods fog x~~~ov-to ing nnio~oorgarai~na~ li%~ ~ ~ n~.~ ~~t~,~oli~io~ ~~o~
~~d blood cell products p~iox to long °t~r~ ~tc~~~g~~
lt~c~aresu~~ ~~ the ~ave~nt cue:
The published lit~r~tux~~ x~port~ the pros~
e~ce o~ ~~~11 numbs~~ o~ ~aicrooxg~xai~~~ in donog blood ~t the tixa~ o~ a~l~.~ction. ~~ ~hoc~n in ~'~ble l, Myh~e et ~1.. have detected the presence o~ ~ro~ 1 tea ~ b~c-~
t~~ci~l strains ~t ~,he~ ti~a~ off' phleboto~ay, pith .~o~,~
strains b~inc~ pr~a~nt at ccneent~°~tion~ g~~~t~r than o~'g~113t~1~s g3eY' '~11. ~lyha.'~ ~t ~l . , °emact~~'iocid~l 2~ Prop~~ti~~ o~ platelet Conc~nt~~t~s,e~ T~~n~~usi~n 14:116 (1~7~).

Table 1 Organisms Isolated From the First 7 mL of Donatedl Blood From Nine Normal Donors Bacteria occurrence Bacterial Con-(N~9) centration S ora/ml) Diptheroid sp. 1 10 colonies/7ml Staph. epid. 6 1-200+

Bacillus sp. 9 1-200+

Micrococcus 1 4 Staph. aureus 4 1-100+

Neisseria sp. 2 1-100+

alpha. strap. 2 2-100+

Staph. sp. 1 1 While: there may be bacteria present at the time of blood collection, the presence of clinically significant numbers of bacteria at the time of trans-fusion is rarely encountered. For many years, this decline in the number of bacteria in donor blood be-tween time of collection and the time of transfusion has been attributed to a sterilizing activity of white blood cells and plasma factors. See, for example, Buchholz et al., "Bacteria Proliferation in Platelet Products Stored at Room Temperature," 285:429 (1971): and Punsalang et al., "Growth of Gram-Positive and Gram-Negative Bacteria in Platelet Concentrates,"
Transfusion 29:596 (1989) The occurrence of transfusion-induced sepsis is considered .less likely with stored red blood cells (which are refrigerated during storage at about 4 de-grees C), compared to platelet concentrates (which are stored at roomy temperature). Still, there~have been reports of sepsis caused by transfusions of refriger-ated red blood cells with the organism Yersinia enterocolitica (which will be referred to as "Yersinia"). Yersinia is a human pathogen that can multiply in blood even during refrigerated storage at WO 92/19355 fCx'/lJS92/03475 - 3 - ~~~~7 l.~t~
~ degrees G. The occurrence of sepsis due to the presence of Yersinia in stored blood products is very rare, with only about twenty-three cases reported in the United States in the last nine years. Still, it would be desirable to com~aletely eliminate its occurrence altogether.
_su_ rmmary ~g ~th~ Invan~ta.on o The invention provides blood processing methods that significantly reduce the presence of mi croorganisms like Yersinia in stored red blood cell products.
The method that embodies the features of the invention collects a blood product containing red blood cells in a first container that forms a part of a sterile, closed blood collection system. The method refrigerates the blood product in the first container to cool the blood product to a temperature of.about 3 to 5 degrees C. The refrigerated blood product is transferred from the first container into a storage ~0 container using a sterile, closed transfer system that includes an inline filter medium comprising a mass of synthetic fibers having an average fiber diameter of about 10 microns or less and a bulk density of about 0.7 gram per cubic centimeter or less. The method stores the filtered blood product in the storage con°
tamer at a temperature of about 3 to 5 degrees C for at least twenty-four hours after filtration for subse-quent transfusion.
The inventors have discovered that, by fol lowing the prescribed method, clinically significant amounts of microorganisms like Yersinia that can be present in 'the red blood cell product at the time of collection can be ~rirtually eliminated. The pre scribed method provides a red blood cell product that is free of clinically significant amounts of microor-ganisms at. the beginning of the storage period. The microorganism-depleted condition persists throughout the storage period up t;o the time of transfusion.
The discovery is surprising and unexpected. At the present time, the inventors do not know exactly why or how the inventic:>n achieves the benefits it does.
It is known that the filter medium used in the prescribed processing method serves to remove essentially all the leukocytes from the whole blood and red blood cell products. Since leukocytes are thought to scavenge microorganisms pre;~ent in stored blood products, conventional wisdorn would expect an increase in the number of microorganisms in the filtered,.leuko-depleted red blood cell product during storage for over twenty-four hours. Surpri;~ingly, the ~_nventors have found the reverse to be true. The filtered red blood cell product remains free of cl_Lnically significant amounts of microorganisms for storage periods well in excess of twenty-four hours, despite also being in a leuko-depleted condition.
According to an aspect of the invention, a method of processing a blood product containing red blood cells to :remove microorganisms prior to long term storage, i~he method comprises the steps of:
collecting the blood product containing red blood cells in <~ first container that forms a part of a sterile, closed blood. collection system including a storage container, a first fluid path that leads into the storage container, the first f-quid path including a dry inline fi:Lter medium. comprising a mass of synthetic fibers hawing an average fiber diameter of about 10 microns o:r less anc:~ a bulk density of about 0.7 gram per cubic centimeter o:r Less, and a second fluid pith that -4a-leads from the star:'age container to the first container and bypas;~es the f ztter medium, adding a storage solution t;o the blood product without wetting the filter medium, before conveyz.ng any of the blood product containing the storage soluticm from the first container into the storage container through the first fluid path and the filter medium, and. before wetting the filter medium, refrigeral~ing the blood product: containing the storage solution :in the fig:gist container together with the closed blood col:Lection s;rstem to cool the blood product containing the storage solution and the dry inline filter medium to a temperature of about 3 to 5 degrees C°, thereby creating a precooled blood product containing the storage solution and <~ precooled dry inline filter medium, only after both the blood product containing the storage solution and the dry inline filter medium have been cooled to a temperature of about 3 to 5 degrees C°., conveying the precaoled blood product containing the storage solution from the first container into the storage container i~hrough the first fluid path and the preceded :Filter me<:~ium t:o remove microorganisms from the precooled blood product, venting air f:rorn the storage container into the first container through the second fluid path that bypasses the filter medium, and after venting the air from the storage container, storing t:he filtered, microorganism--depleted blood product containing the l~torage solution in the storage container at a temperature of about 3 to 5 degrees C°.
for a period that exceeds twenty-four hours after f iltratio:n.

-4b-In accordance with another aspect of the invention; use of the filtered microorganism-depleted blood product at the end of the storage period for transfusion.
Other features and advantages of the invention will become apparent upon review of the following description, drawings, and appended claims.
Brief Description of the Drawings:
Fig. 1 is a schematic view of a blood collection system that includes a blood processing assembly <~nd a blood transfer assembly that embody the features of the invention;
Fig. 2 is a schematic view of the system shown in Fig. 1, with the blood transfer assembly attached to the blood processing assembly for the purpose of removing undesired matter from the blood cells;
Fig. 3 is a schematic view of the system 9W0 92/193~~ PC'~'/r.7S92/03~475 ~ ~;1~ j:_J~_J
shown in Fig. 1, ~rith the blood cells, now substan-tially free of undesired matte:, ready for long term storage; and Fig. 4 is an enlarged side sectional view of 5 the sterile connection devices associated ~~rith the system shown in Fig. 1.
D~ecri~tion ~f tYxa Preferred ~nbodimantse A blood collection system l0 is shac~n in Fig. 1. The system 10 comprises a blood collection, processing and storage assembly 12 and a transfer as sembly 14.
In the illustrated and preferred embodiment shown in Fig. 1, the transfer assembly 14 comprises an initially separate subassembly not joined to the blood processing assembly 12. It should be appreciated, however, fleet the transfer asse7ably 14 can be made as an integral part of the processing assembly 12.
The blood collectio~a assembly 12 comprises a multiple blood bag system having a primary bag or container 16 and one or more integrally attached transfer bags or containers 18 and 20. In use, the primary bag 16 (which is typically also called a donor bag) receives whole blood from a donor through inte-grally attached donor tubing 22 that carries an phle-botomy needle 24. A suitable anticoagulant A is con-tained in the primary bag 16.
~. satellite bag 26 is attached to the prima-ry bag 16 by integrally attached tubing 28. The sat-ellite bag 26 contains a suitable storage solution S
for red blood cells. One such solution is disclosed in Grode et a1 U.S. Fatent 4,267,269.
The transfer bags 18 and 20 are attached to the primary bag 16 by integrally attached transfer tubing 30 and 32. 'Phe transfer bags 18 and 20 are in-tended to receive the platelet and plasma blood compo-WO 92/19355 CA 02086516 2001-08-15 p~'/LTS92/03475 nents for processing. The first transfer bag 18 ulti mately serves as the storage container for the plate let concentrate, and the second transfer bag 20 ulti mately serves as the storage container for the plate s let-poor plasma.
All of the bags and tubing associated with the processing assembly 12 can be made from conven-tional approved medical grade plastic materials, such as polyvinyl chloride plasticized with di-2-:LO ethylhexylphthala.te (DEHP). Alternatively, the first transfer container 18, which is intended to store the platelet concentrate, can be made of polyolefin mate-rial (as disclosed in Gajewski et al U.S. Patent 4,140,162) or a polyvinyl chloride material :l5 plasticized with tri-2-ethylhexyl trimellitate (TEHTHj. These materials, when compared to DEHP-plasticized polyvinyl chloride materials, have greater gas permeability 'that is beneficial for platelet stor-age.
a0 The blood collection assembly 12, once ster-ilized, constitutes a sterile, "closed" system, as judged by the applicable standards in the United States.
Whole blood is collected and then separated 25 into its various. therapeutic components within the assembly 12. These therapeutic components are typi-cally red blood cells, plasma, and platelets. In use, the collected whole blood is centrifugally separated within the primary bag 16 into red blood cells and 30 platelet-rich plasma. The platelet-rich plasma is transferred by. cc>nventional techniques into the first transfer bag 18 leaving the red blood cells in the primary bag. The transfer bags 18 and 20 are detached in a sterile fashion using a conventional heat sealing 35 device (for example, the Hematron~ dielectric sealer W~ 92/19355 ~CI'/11~92/t)3~475 a a a ~.~ r -; -~ , 1~ U '.f ~:j r. V
sold by Baxter ~Iealthcare Gorpora-tion) , which forms a hermetic, snap-apart seal in the tubing 30 (this seal is schematically shown by an "x"' in Figs. 2 and 3).
The red blood cell s>torage solution S is transferred into the primary container 15,~ and the satellite bag 26 is then disconnected using the snapm apart seal "x" (as shown in Fig. 2). The donor tubing 22 is also sealed and disconnected in the same fashion (as also shown in F'ig. 2).
The platelet-rich plasma can undergo subset quent centrifugal separation within the first 'transfer bag 18 into platelet concentrate and platelet-poor plasma. The platelet-poor plasma is transferred into the second transfer bag 20, leaving the platelet con--centrate in the first transfer bag 18. The transfer bags 18 and 20 are then separated by the snap~apart seals a'x°' in the tubing 32 (as shown in Fig. 2) for subsequent storage of the collected components.
The transfer assembly 14 includes a storage container 3~ and an associated fluid flow path 36. An optional air vent path 38 is also provided (shown in phantam lines in ~'ig. 1 to 3). The transfer assembly 14 includes a conventional roller clamp ~6 associated with the flow path 36. The transfer assembly 1~ also includes a conventional roller clamp ~7 associated with the optional air vent path 38.
The transfer container 34 and fluid paths 36 and 38 are all made of low cos°t medical grade plastic materials, such as polyvinyl chloride plasticised with DEHP.
The fluid path 3E includes an inline filtra~
tion device 40 for separating undesired matter from refrigerated whole blood and other refrigerated blood products that contain red blood cells.
In the illustrated embodiment, the filtra-_8_ tion device 40 includes a housing 42 containing a filtration medium 44. The filtration medium 44 comprises a mass of synthetic fibers having a average fiber diameter of about. a.0 microns or less and a bulk density of about 0.7 gram per r_ubic centimeter or less.
Filtration mediums of this type are described in Takenaka et al. U.;3. Patent 4,330,410 and Watanabe et al U.S.
Patent 4,'701,267. Filters having filter med,~ums of this type are <also commercially available and sold by the Fenwal Division of Baxter Healthcare Corporation under the tradename Sepacell R-500 Leukodepletion Filters.
In the illust~:-ated and preferred embodiment, a connection assembly 48 is associated with the initially separate blood collection and transfer assemblies 12 and 14. The connection assembly 48 permits selective attachment of the transfer assembly 14 to the blood collection assembly 12. Once attached with the flow control device 46 c:~pened (and optional flow control device 47 closed? (as shown in Fig. 2?, red blood cells can be conveyed from the primary container 16 through the flow path 36 and filtration device 40 into the storage container 34.
Whip=_ the two assemblies 12 and 14 are still attached 'together, the flow contro-'~ device 46 can be closed and optiona:L f:Low control device 47 opened to vent entrapped air from the storage container 34 through the vent path 38 into th.e primary container 16.
The storage container 34 :LS then detached from the transfer assembly 14, as shown in Fig. 3. The filtered red blood cell. blood product is stored in the separated container 34.
In t:he illust:rat.ed and preferred embodiment, the filtration assembly 14, once sterilized, comprises a sterile, "closed" system (like the processing and storage assembly 12), as judged by the applicable United States standards. In this arrangement, the connection assembly 48 serves to ;attach and det=ach the collection and filtration assembly in a manner that preserves the sterile integrity of the closed systems 12 and 14.
More particularl;r, the connection assembly 48 comprises two mating sterile connection devices (designated 66a and 66b). The devices 66~a and 66b (see also Fig. 4) are described in Granzow et a]. U.~3. Patents 4,:157,723 and 4,265,280. One device 66a is carried by tubing 68 attached to the primary bag 16. The other device 66b is carried at the end 54 of the flow path 36 of the transfer' assembly 14.
As shown in Fic~. 4, the sterile connection devices 66a and 66b ea~~h generally includes a housing 70 having a normally closed, meltable wall 72 made of a radiant energjT absorbing material. 'The housings 70 are joined together with mating bayonet-type couplers 74a and 74b, with the walls 72 placed in facing con-tact. When connected and exposed to radiant energy, the walls 72 melt at temperatures that result in the destruction of bacteria, while at the same time opening a fluid path between the connected housings 70. In this Figure, as shown i:n more detail, the end of the flow path is tubing end 54 and tubing 50 provides the aforementioned flow path 36.
The devices 66a <~nd 66b normally close the associated assemblies 12 and 14 from communication. with the atmosphere and are opened in conjunction with an active sterilization step which serves to ~.t.erilize the regions adjacent to the interconnecting fluid path as the fluid path is being formed.
These devices 66a and 66b also hermetically seal the interconnecting fluid path at the time it is formed. The use of these W~ ~32/1935~ PC'T/~.J592/03A75 _ sterile connection devices 66a arid 66b assures a prob-ability of non-sterility that exceeds one in a mil-lion. The devices 66a and 66b thus serve to connect the two assemblies 12 and 14 wit',hout compromising the sterile integrity of either. ' Alternately, the connection assembly 4a can comprise the sterile connecting system disclosed in Spencer U.S. Patent 4,412,535 (not shown). In this arrangement, this system forms a molten seal between the transfer tubing 30 of the primary bag 16 with the tubing end portion 54 of the filtration assembly 14.
t3nce cooled, a sterile weld is formed.
It is known that the just described filtra tion medium 44 is suited for the removal of white blood cells and platelets from refrigerated whole blood and red blood cells. The inventors have discov-ered that the filtration medium 44 also removes micro-organisms like Yersinia from refrigerated red blood cell products prior to long term storage. This microorganism-depleted condition the invention pro-vides persists during long term storage of ~°ed blood cell products at temperatures of about 3 to 5 degrees for periods well over twenty-four hours.
In use, whole blood is collected in the do nor bag 16, which forms a part of the blood collection assembly 12. After removal of the platelet-rich plas ma and detachment of the transfer bags (see Fig. 2), the red blood cells remain in the donor bag 16. The storage solution S is conveyed from the bag 26 into the donor bag 1.6, and the bag 26 is disconnected. The resulting red blood cell product is refrigerated in the donor bag 26 to cool the product to a temperature of about 3 to 5 degrees C.
Following refrigeration, the donor bag 16 is attached to the transfer assembly 14 using the associ-'W~ 9Z/i~D355 ~'C'x'/LJ592/03475 °' 11 " ~l "~ ~ a ~l ~. ;=i ated sterile connection devices 66a and 66b. The roller clamp 46 is opened, and the roller clamp 47 is closed.
As shown in Fig. 2, the donor bag 16 is lifted above the storage bag 34, and the refrigerated red blood cells are conveyed by gravity flow from the donor bag 16 through the fluid path 36 and filtration device 40 and into the storage bag 34. Microorganisms arc removed from the red blood cells by the filtration l0 devise 40. T~eukocytes are also removed in the pro-cess.
Should air be trapped in the storage bag 34, it may be necessary to transfer the air through the vent path 3~ into the donor bag 16 in the manner pre viously described.
The storage bag 34 is then separated from the transfer assembly 14. This is accomplished by forming snap-apart seals a'xaa in the tubing 36 of the transfer assembly 14 (as Fig. 3 shows).
The leukocyte-depleted red blood cell prod-uct, now substantially free of microorganisms like 1'ersinia, is stored in the storage bag 34 at temperatures of from 3 to 5 degrees ~ for a period over twenty-four hours before transfusing.
The following example demonstrates the sur-prising and unexpected results of the invention.
~P~E
Units of whole blood were collected from different donors in separate sterile, closed multiple blood bag sy~>tems, like that shown in Fig. 1. Within thirty minutes of collection, test units were prepared using aseptic: conditions. Rach test unit was made by po~ling two units of d3B~-compatible whole blood from .
two different donors. These test units are identified as Pools A tlZrough ~ in the Result Tables 2 to 5 be-v~o 9xeb93ss ~~reu~~xeo~a~s ~6~~~~.~! ~ - 12 -low.
The Pools of whole blood were inoculated with Yersinia at four different s.noculation levels: at abaiat 7. organism per ml (Pools A, B, and C); at about 2 to 3 organisms per ml (Pools p, E, and F);~at about 30 organisms per ml (Pools G, H, and I); and at about 135 organisms per ml (Pools J, K, and 7G). Each inoculated pooled test unit was mixed well.
It is believed that the inoculation levels Of below 10 organisms par Ial. represent amounts of Yersinia that could be present in the blood of an asymptomatic or otherwise apparently healthy prospec tive donor. Uue to the lack of atypica7_ symptoms, these persons would be permitted to loin the pool of prospective blood donors. Therefore, it is possible that blood containing Yersinia at these asymptomatic levels could be collected.
It is believed that the higher inoculation levels of more than 10 organisms per ml represent amounts of Yersinia that, if present in the blood of a prospective donor, would manifest itself as fever or other atypical physical symptoms. The atypical symp-toms would be detected during routine pre-collection examination, eliminating these symptomatic parsons from the pool of prospective blood donors. Therefore, it is unlikely that blood containing Yersinia at these high, symptomatic levels would be collected.
After mixing, each inoculated Pool of whole blood was broken into single unit pairs (referred to as Units A and B in the Result Tables below). Each unit pair was sampled for quantitative culture (as , shown in the "Pre-Hold°° column of the Results Table).
The inoculated Unit pairs for Pool were he7.d for seven , hours at room temperature and again sampled for quan--titative culture (as shown in the '°Pst-Hold" column of W~ 92/19359 PC,'T1U592/03~d75 ~3 -the Results Table).
Each inoculated Unit pair of whole blood was processed into platelet-rich plasma and red blood cells in the manner described in this Specification.
The red blood cell storage solution described in Grode et al U.S. Patent 4,267,269 was added to the red blood cells in each Unit pair.
The resulting red blood cell product for sash inoculated Unit pair were refrigerated for 18 to 24 hours to a temperature of 4 degrees C. The red blood cells for each inoculated Unit pair where then sampled for quantitative culture (as shown in the "PreFilt Day 1'° column of the Results Table).
Unit A of each red blood cell product pair Z5 was transferred into another storage container via the inline filtration device described above. Red blood cell product Unit A were sampled after filtration for quantitative culture (as shown in the "PstFalt Day 1"
column of the Results Table). The Units A were then stored at 3 to 5 degrees C. Quantitative cultures were obtained at the end of 7, 14, 21, 28, 35, and 42 days of storage (as shown in the Day columns in the Results Table).
unit B of each red blood cell product pair was stored as a control without undergaing filtration at 3 to 5 degrees C. Quantitative cultures were ob tained at the end of 7, 14, 21, 28, 35, and 42 days of storage (as shown in the Day columns in the Result Tables).
The results of the study are summarized in the follawing Results Tables 2 to 5.

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The foregoing Results Tables demonstrate that clinically significant amounts of microorganisms like Yersinia present in the red blood cell product at the time of collection can be either eliminated or significantly reduced by filtration in accordance with the invention.
At inoculation levels representative of asymptomatic or otherwise apparently healthy donors (see Results Tables 2 and 3), the microorganism-de-plated condition created in accordance with the inven-tion at the outset of the storage period persists for extended storage intervals of at least 42 days. Even at a higher symptomatic inoculation level of about 30 organisms per ml. (Results Table 4), the microorgan-ism-depleted condition is observed after 2a days of post-filtration storage. At these inoculation levels, red blood cell products that were not filtered in accordance with the invention exhibited clinically , significant amounts of Yersinia organisms by about the seventh day of storage and confluent growths of Yersinia organisms by the twenty-first day of starage.
Even at Yersinia inoculation levels that are more than 10 times that would be present in asymptomatic or otherwise apparently healthy donors (see Result Table 5), the beneficial results of the invention are observed during storage periods of at least seven days.
Various features of the invention are set forth in the fOllOwlng ClalmS.

Claims (2)

1. A method of processing a blood product containing red blood cells to remove microorganisms prior to long term storage, the method comprising the steps of:
collecting them blood product containing red blood cells in a first container that forms a part of a sterile, closed blood collection system including a storage container, a first fluid path that leads into the storage container, the first fluid path including a dry inline filter medium comprising a mass of synthetic fibers having an average fiber diameter of about 10 microns or less and a bulk density of about 0.7 gram per cubic centimeter or less, and a second fluid path that leads from the storage container to the first container and bypasses the fitter medium, adding a storage solution to the blood product without wetting the filter medium, before conveying any of the blood product containing the storage solution from the first container into the storage container through the first fluid path and the filter medium, and before wetting the filter medium, refrigerating the blood product containing the storage solution in the fiat container together with the closed blood collection system to cool the blood product containing the storage solution and the dry inline filter medium to a temperature of about 3 to 5 degrees C.., thereby creating a precooled blood product containing the storage solution and a precooled dry inline filter medium, only after both the blood product containing the storage solution and the dry inline filter medium have been cooled to a temperature of about 3 to 5 degrees C°., conveying the precooled blood product containing the storage solution from tine first container into the storage container through the first fluid path and the precooled filter medium to remove microorganisms from the precooled blood product, venting air from the storage container into the first container through the second fluid path that bypasses the filter medium, and after venting the air from the storage container, storing the filtered, microorganism-depleted blood product containing the storage solution in the storage container at a temperature of about 3 to 5 degrees C°.
for a period that exceeds twenty-four hours after filtration.

2. Use of the filtered microorganism-depleted blood product at the end of the storage period for transfusion.