CN103250694A

CN103250694A - Composition and method for storing erythrocyte

Info

Publication number: CN103250694A
Application number: CN2013100691113A
Authority: CN
Inventors: J.R.赫斯; T.G.格林沃尔特
Original assignee: University of Cincinnati
Current assignee: University of Cincinnati
Priority date: 2005-02-17
Filing date: 2005-02-17
Publication date: 2013-08-21

Abstract

The invention provides a method for storing erythrocyte. A composition for storing erythrocyte is basically composed of the following substances: adenine, dextrose, at least a non-metabolizable membrane protective agent sugar and a specifically-defined pH buffer system. The invention also provides an improved method for storing erythrocyte and a method for increasing viability, membrane preservability and restorability of stored erythrocyte and inhibiting apoptosis, hemolysis and clearance rate after reinfusion, wherein the novel composition is used.

Description

The composition of storage of red blood cells and method

The application is to be on February 17th, 2005 applying date, and application number is dividing an application of application for a patent for invention 200580049506.0, that denomination of invention is identical with the present invention.

John?R.Hess

Tibor?J.Greenwalt

About the research of federation's patronage or the statement of development

This part of work is carried out under septic yanks and about DAMD17-95-C-5029, and U.S. government can enjoy owner's equity at this.

Background of invention

The present invention relates generally to composition and the method relevant with storage of red blood cells (RBCs).Be particularly related to improve composition that RBC stores and method with and use.

Store with preserve red blood cell (RBCs) be used for being filled into again thereafter ability in patient's body be a kind of relative recently put into practice pioneer's development in science and technology as modern surgery.This being kept on the science is thorny, and realizes long term storage and obtain high-quality perfusion more also more and more with erythrocytic step.In case collect red blood cell from the donor, they just begin dead because they can condense, hungry, lose ATP, 2,3-DPG, film surface area and integrality, and hemochrome (Hb).Rous﹠amp; Turner is at first successfully demonstrated in 1917 storage of whole blood of 1916 and Robertson.Afterwards, acid citrate dextrose (ACD, 1943), it contains as the citrate of anticoagulant with as the unique nutriment dextrose that is utilized by red blood cell, and citrate phosphate dextrose solution (CPD, 1957), wherein added phosphate as the metabolism source and be used for film and fixed, approved to be used for storage of whole blood 21 days.Contain the whole blood that CPD (CPDA-I, 1979) is introduced into subsequently and energy will be stored of adenine and extended pot life to 5 week of concentrated RBCs (packed RBCs).

At first, storage component is designed to acid to prevent during the glucose heat sterilization in the production stage in the end by caramelization.In the 1950's, adenine is found can be as additive and the adenine loss that replaces deaminizing to cause.In the seventies in 20th century, for blood platelet and production blood plasma derivant, begin to expect from the whole blood of collecting, to remove blood plasma.Yet this rate of recovery that can cause gained " to concentrate RBC (packed RBC) " descends.

In order to prevent this from occurring, to have developed conduct known in the art and added the composition of solution (AS) with compensation capacity, nutrient and other useful RBC stabilizing agent.Be necessary adjusting especially so that it meets the needs of RBCs for the protection of the interpolation liquid composite that from whole blood, separates back red blood cell (RBCs).Developed in 1981 specific interpolation solution with the extended pot life of RBC to 6 weeks.Yet the red blood cell (RBCs) that is stored in this solution stable degeneration but occurred after about 6 weeks, and all to be determined be useless because this cell of surviving in 24 hours the circulation after being filled into people's donor again has 75%.Have been found that during continuous refrigerated storage, glucose is consumed with speed decrescence, because metabolic waste, namely lactic acid and hydrionic concentration are increasing.The decline of this glucose metabolism speed causes adenosine triphosphate (ATP) consume, and it is directly related with the recovery (recovery) of RBCs that this returns to circulation time at cell.For example Adsol.RTM (AS-I), Nutricel RTM (AS-3), Optisol RTM (AS-5) and ErythroSol RTM are used for prolongation down in the storage period of 1-6 ℃ of following RBCs to have designed interpolation solution.At all these ASs of U.S.'s approval, AS-I, AS-3 and AS-5 comprise salt solution, adenine, glucose and some citrates and/or mannitol as " film protectant " recently.AS-3 also comprises sodium dihydrogen phosphate.The ASs of each U.S.'s approval meets the permission requirement of storing RBC6 week, but can not store for 7 weeks.At present the RBC of approval adds liquid composite and stores and just be developed before damage (be defined herein as RBCs store in to the summation of survival and/or function restriction) is a kind of apoptosis process knowing RBC.

Nearly all collection whole blood all is made into component at present, and the RBC part is stored with the form that concentrates RBCs.Add in the solution system for blood is incorporated into, we concentrate RBCs by freezing, remove blood plasma so that RBCs accounts for 80% volume, sterilely add 100ml then and add solution.The RBC volume of gained suspension partly accounts for 55%.The RBCs that is stored in the interpolation solution of general FD A-approval only can be stored for 6 weeks, had in 24 hours the body to accept recovery.

Recovery time be can to accept in order being increased in to store in the body that is filled into RBCs in patient's body after a period of time again, interpolation solution and storage procedures to have been attempted to have improved.In " the research-7 that red blood cell is preserved.Improved ammonium phosphate adds the interior and in vitro study of body of solution " in, Greenwalt etc., Vox.Sang.65:87-94 (1993), the author determines, experimental interpolation solution (being appointed as EAS-2), it contains (mM): 20NH ₄Cl, 30Na ₂HPO ₄, 2 adenines, 110 dextroses, 55 mannitols, under pH7.15, form, can be used for the shelf life of prolonged human RBCs, from the standard in present 5-6 week to the improvement standard that is increased to 8-9 week.Yet, be stored in concentrated RBCs among the EAS-2 and can not directly inject and remove supernatant through washing step before need being used in infusion, there is ammonium because add in the solution.

In " the research-8 that red blood cell is preserved.Erythrocytic liquid storage the in containing the interpolation solution of glycerine " in, Vox.Sang.67:139-143 (1994), Greenwalt etc., having put down in writing can be at the interpolation solution (being appointed as EAS-25) of nine weeks back recovery, 73% Red Blood Cells Concentrate.Yet the RBC unit of gained comprises about 1% glycerine, and this human body infusion for heavy dose is unsafe.

In " 4 ℃ of downward leptocytes store ", Transfus.Sci.15:105-115 (1994), Meryman etc. have proved to be stored in the very light suspension to exist 27 all RBCs that acceptable viability is arranged with low hematocrit value.Yet the suspension of this storage RBC can not be accepted because their potassium and ammonia content height and RBCs volume part are low by direct infusion.Being used for storing 200mLRBC and needing to produce the 5L solution of surveying beneficial effect is infeasible clinically.

With regard to can go through and commercially available product with regard to, the interpolation solution of U.S.'s approval at present is 6 weeks of onset only, on average restore about 80%.Two kinds of Europe approval are at present added solution about 7 weeks of onset, and (ErythroSol from Baxter Healthcare, La Chatre is France) with 75% (PAGGS mannitol from Maco Pharma) on average to restore 77%.The new soln expection of Kurup eta1. (Vox Sang2003:85:253-261) record at present may have shorter storage period, because ATP concentration is low.

At deficiency of the prior art, the present inventor has developed the alkalescence experiment that contains low volume sodium hydrogen phosphate and has added solution (EASs), it can partly neutralize and collect blood to for example effect of CPD (citrate-phosphate-dextrose) of acid anticoagulant solution, and show that these EASs can improve RBC ATP concentration, reduce haemolysis, and reduce the metamorphosis of RBC film and loss (referring to United States Patent (USP) 6,150,085 and 6,447,987, Hess and Greenwalt all introduces its disclosure in this mode by reference).Various EASs support the storage period in 9 to 12 weeks.Although these EASs have produced higher results of property, they comprise sodium chloride and are made into big relatively volume causes and is stored RBC and diluted more, has just increased the danger that blood is diluted like this when being infused into patient's acceptor.In addition, depositing of sodium chloride limited buffer salt and phosphatic meltage makes this system can keep the volume of expection.

As higher time requirement but also when being the time requirement that is interrupted, for example during war, and needing discontinuously, dispersedly in the geographic area of infused blood, the period of storage that improves RBC just seems extremely important.In fact, suppose it is reported that present loss level is because RBCs had just spent the safe storage phase in the past in practical application usually, the safe storage time of therefore improving RBCs is exactly the problem that people generally are concerned about at present.

Therefore, need be at the conventional RBC storage component that can keep or improve RBC recovery and performance in the solution that adds of low volume.Still have in blood storage and infusion art and improve the demand that RBC stores, this can make period of storage prolong, and the recovery percentage is higher, and has improved the physiological function of infusion RBC.Therefore, still need to improve composition and the production method that RBC stores.Also need to add composition, this composition can make the RBC suspension that adds said composition directly be infused in the human body, and can allow active RBCs that acceptable restoration is arranged behind infusion, wherein active RBCs has the physiological function of enhancing and has reduced by the clearance rate in the infusion patient circulation.

The invention summary

Therefore, the invention provides to be suitable for storing and preserve and be collected erythrocytic new composition.The present inventor is surprised to find that, from this composition, remove the high-performance composition of the pH regulating system that sodium chloride (before this be considered to correctly to prepare storage component necessary) can be provided for strengthening basically, this all is useful to storage of red blood cells and the erythrocytic integrality of infusion more subsequently and physiological function character, and in view of the prolongation of time, RBCs just can keep meeting restoration and the hemolysis levels of relevant laws and regulations when storing.In addition, the present composition has kept preferable performance under conventional volume, and this makes them be particularly suitable for storage of red blood cells, wherein red blood cell can be repeatedly or a large amount of infusion give the patient.

One embodiment of the invention provide and have been used at about 1 composition to about 6 ℃ of following storage of red blood cells.Said composition mainly is made up of following material: adenine, dextrose, at least one nonmetabolizable film protectant sugar and pH buffer system.The pH buffer system contains sodium bicarbonate and sodium hydrogen phosphate and is that about 8 to about 9 amount exists enough to make pH.Said composition can keep the pH of red blood cell (RBC) suspension, wherein said composition is enough to set up in red blood cell at lay up period and to keep the amount of following molecular balance to be added in the suspension, described balance is that a kind of inhibition is from 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and help glucolytic balance has produced the net profit of adenosine triphosphate (ATP) thus in the molecular balance of lay up period.Another embodiment of the invention provides the composition that is substantially free of sodium chloride.

More particular embodiment of the present invention provides specific component and content thereof, the pH of Morie osmolarity scope and composition.Other specific embodiments is at making erythrocytic pH remain on the present composition in the special value scope.

Another embodiment of the invention provides the red cell suspension that contains the present composition.

The method embodiment also is provided.Such embodiment is at the method for preserving red blood cell (RBCs) at lay up period.This method comprises: (a) will contain the RBCs that need remain to be stored and the whole blood sample that is collected of blood plasma and mix with anticoagulant solution, and form the suspension of collected whole blood thus; (b) suspension of collected whole blood is handled to remove blood plasma and concentrated RBCs, form thus and concentrate RBCs; (c) Packed RBC s is mixed with a certain amount of composition, this amount is enough to form has an appointment 35% to the RBCs suspension of about 70% volume RBCs; (d) cooling RBCs suspension is to about 1 to about 6 ℃; And the RBCs suspension of (e) storing cooling according to the standard storage step.Said composition mainly is made up of following material: adenine; Dextrose; At least one nonmetabolizable film protectant sugar; With the pH buffer system.This pH buffer system comprises sodium bicarbonate and sodium hydrogen phosphate and is that about 8 to about 9 amount exists with the pH that enough makes composition.Said composition can keep the pH of red blood cell (RBC) suspension, wherein said composition is enough to set up in red blood cell at lay up period and to keep the amount of molecular balance to be added in the suspension, this balance is that a kind of inhibition is from 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and help glucolytic balance has produced the net profit of adenosine triphosphate (ATP) thus in the molecular balance of lay up period.More particular embodiment also is provided.

Other embodiments also are provided, it is at using the present composition to keep and suppress the method for RBC apoptosis with the film that improves red blood cell (RBC) in storage period, with the fragility that reduces red blood cell storage period (RBC) and suppress RBC haemolysis, and improve the viability enter storage period subsequently and to be infused into back red blood cell (RBC) in the patient's body that needs this infusion, and reduce behind patient's infusion the clearance rate to RBC.

The composition and the RBC suspension that generate according to the present invention provide a kind of like this storage period for RBCs, namely in whole storage period, the RBCs that sufficient therapeutic dose is arranged is recoverable and can directly be infused in patient's body and need not with setting up and verifying that the known standard method of RBCs infusion handles.

These and other embodiment of the present invention and various aspects will be by sketching, the detailed description of the following preferred embodiment that provides and embodiment being understood more fully with reference to the accompanying drawings.

The accompanying drawing tube is stated

Fig. 1: be the graphic representation of mixing (pooling) result of study of expression RBCs storage effect, with the time (week) be function, relate to 4 different interpolation liquid composites: 1) AS-3, volume 110mL, (◇-); EAS-61, volume 170mL (◆-); EAS-78,170mL, (●-); And EAS-81,110mL, (zero-).Contain the composition of bicarbonate, represent with circle, compare with the equal-volume composition (representing with rhombus) of potassium-containing hydrogen salt not, produced higher ATP concentration, shown in figure A.These compositions also relate to higher lactate concentration (figure D), the outer and interior pH of born of the same parents (figure E and F) of higher born of the same parents, and higher bicarbonate and PCO ₂Concentration (figure E and F).The composition that volume is bigger is represented with solid figure, and the hematocrit value reduction that haemolysis reduces and stores is described, this is illustrated in figure B and C respectively.

Fig. 2: explanation will be stored in 6 weeks (n=6) among the EAS-81, the RBCs in 8 weeks (n=6) is infused into the red blood cell restoration in vivo of sampling in back 24 hours in the subject again among the EAS-81, compare with contrast in the past, the permission of AS-3 experiment is announced in 1985 by Simon etc.These two researchs have all been used ⁵¹Cr list-labeling acts (single-label method).Licensure?study

The description of preferred embodiment

The present invention relates to store relevant composition and method with red blood cell (RBC).Be particularly related to new interpolation liquid composite and the method relevant with storing RBCs, wherein RBCs separates from whole blood, wherein whole blood is in citrate phosphate dextrose (CPD) solution, its variant, collect in the dual dextrose of citric acid phosphoric acid salt (CP2D) solution, or in acid citrate dextrose (ACD) or similar solution, collect by extracting (Aphaeresis) (from patient or donor, removing whole blood).

With regard to purpose of the present invention, term " recovery " refers to after being filled into initial people's donor more as used herein, keeps 24 hours storage RBCs part in circulation.

" chloride " refers to the anion chloride as used herein.Therefore, term " chloride " comprises anion chloride and salt form thereof, for example can be formed by chloride anion and physiologically acceptable cation.Term " chloride " does not comprise that chlorine atom wherein is to receive the compound on the carbon atom in the organic molecule for example with covalent bond.

Phrase " physiologically acceptable buffer " refers to the buffer that can produce cation and anion as used herein, and wherein this ion generally is found in people's blood, blood plasma or the serum, maybe can be tolerated when introducing human body.Suitable cation comprises proton, ammonium cation and metal cation.Suitable metal cation includes but not limited to sodium, potassium, calcium and magnesium cation form, wherein preferred sodium and potassium, more preferably sodium.Ammonium cation, i.e. formula R ₄N ⁺Compound, wherein R is hydrogen or organic group, so long as physiologically acceptablely just can be used.In preferred embodiments, cation is selected from hydrogen (being proton), sodium, potassium, calcium, magnesium and combination thereof." buffer " refers to the reagent that can adjust and regulate composition pH as used herein.

The present composition disclosed herein is moisture, and namely they form in water.The preferred water of the present invention is processed in order to be substantially free of pyrogen (i.e. sterilization).

" mEq/L " refers to the concentration with the special component (solute) of the proportional existence of water amount as used herein.More specifically be that mEq/L refers to the milliequivalent number of solute in every premium on currency.Every liter milliequivalent is calculated by the following method, and every liter of molal quantity of solute and charge species (group) number of per molecule solute are multiplied each other, and multiply by the factor 1,000 then.

One embodiment of the invention provide and have been used at about 1 Aquo-composition to about 6 ℃ of following storage of red blood cells.Said composition mainly is made up of following material: adenine; Dextrose; At least one nonmetabolizable film protectant sugar and pH buffer system.This pH buffer system contains the composition that can accept buffer on the physiology, and must comprise the reagent that at least one can provide bicarbonate anion, and at least one can provide the reagent of phosphate radical anion can provide the reagent of sodium cation with at least one.The present invention expection, single buffer salt can satisfy these in requiring more than one.

Known in erythrocytic preservation field, the ATP concentration in the red cell suspension system is with healthy maximally related.Red blood cell by the final lactate that produces of the sugar decomposition conversion of d-glucose (dextrose), produces ATP through glycolysis thus.Therefore, the Lactated concentration curve synthetic good indicator of ATP just.The reserve force of guard system is not how, and the erythrocytic life-span all is limited and the red blood cell collected comprises the normal distribution of red blood cell age and close to natural death.Owing to do not have new RBCs to enter saved system, therefore to just having produced restriction maximum storage period, wherein should maximum will provide the necessary recovery percentage in perfusion back again storage period.Therefore, although producing the ability of ATP on the whole, system will descend in time; But generally can see add to add behind the liquid be at first raise because it provides the nutrient higher than nature concentration, and RBC experienced " swelling " at first, this also with ATP utilize descend relevant.

Be not limited to any theory, we find, in the time of in being stored in according to interpolation solution of the present invention, the volume increase of nutrient solution can make substrate quantity increase in order to can be transferred with acceptable concentration, and provides the solute that is used for the dilution metabolic waste to reduce the feedback inhibition of glucose metabolism thus.Further supposition, another feature that the present invention adds solution are that the RBCs that they can produce swelling at first reduces gradually at the lay up period erythrocyte volume subsequently.This process is called as " reduction of modulability volume ".Suppose in this process, the activity inhibited of tyrosine phosphatase among the RBC, perhaps tyrosine kinase is activated.Proved that these two enzymes are very a large amount of (Zipser, Y.and Kosower, N.S. (1996) Biochem.J.314:881 in the film of these cells; (1997) FASEB such as Mallozzi C. J.11:1281).The pure phosphoric acidization of band 3 protein will cause from it and with discharging intracytoplasmic phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase (Harrison, M.L. etc. (1991) J.Biol.Chem.266:4106 3 the bonding state on the expection RBC film; Cossins, A.R.and Gibson J.S. (1997) J.Exper.Biol.200:343; Low, P.S. etc. (1993) J.Biol.Chem.268:14627; Low, P.S. etc. (1995) Protoplasma184:1961).The validity of these three enzymes will be expected the glucose metabolism that can improve RBC in the Embden-Meyerhof-Parnaspathway approach, promote the concentration of ATP among the gentle RBCs of synthetic water of ATP thus.Therefore, the purpose of preparation interpolation liquid composite is that synthesizing in the long as far as possible time of ATP remained on the higher speed.

The present inventor finds, synthetic to remain on maximized key be that pH with RBC in the born of the same parents remains on as far as possible near on 7.2 the level with the ATP of system, reaches as far as possible and needn't reach fully.At lay up period, the characteristics of ATP concentration are to remain on the certain level or or even descend then in the early stage rising of storing.When RBC ATP concentration drops to 2 μ mol/g Hb when following, the recovery of RBC generally just is lower than 75%.RBC is storing wall losses 2 early, 3-DPG.The characteristics of initial concentration are to be about 15 μ mol/g Hb or about 1.1 μ mol/g Hb.In 7 to 10 days, concentration generally drops to 1/10th of initial amount.The synthesis rate of 2,3-DPG is the function of pH, occurs greater than 7.2 o'clock but just decline when being lower than this pH at pH.Improve the synthetic trial of 2,3-DPG by the pH that increases storage system and be limited because follow each 2, what the formation of 3-DPG molecule all can one mole one mole ground loss ATP is synthetic.Therefore, increase 2 of RBC, 3-DPG concentration, this is former just to be considered to desired, in fact is the period of storage of tending to reduce RBC.

More the environment of slant acidity has reduced the metabolism of RBC.7.2 pH be crucial, wherein triggered a kind of mechanism, it is known to be that the Rappaport bypass is (referring to " alkaline CPD and red blood cell 2, the preservation of 3-DPG " (2002) Transfusion such as Hess, 42:747-752, at this by with reference to all introducing), wherein 2,3-DPG is by 1,3-DPG is synthetic, consumed the required phosphate of synthetic ATP, also had the glycolysis step in addition, this step has generated two ATPs that produced by glycolysis.The pure effect of system is loss ATP.Can remain on below 7.2 as pH in the carpogonium, this bypass is just closed effectively so, and ATP is synthetic also just to be maximized.Under nature, effect has to a certain degree been brought into play in this bypass, some 2, the generation of 3-DPG and to keep with respect to other cell event be important.Yet the present inventor finds that when storing in Yi Wai the environment in vivo, for the purpose to the red blood cell protection, expectation can minimize this bypass effect.

Therefore, the embodiment of the present composition just provides the pH that exists with the amount that enough meets the following conditions buffer system: wherein said composition is set up and is kept the amount of following molecular balance to be added in the suspension being enough at lay up period in red blood cell, above-mentioned balance is that a kind of inhibition is from 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and help glucolytic balance has produced the net profit of adenosine triphosphate (ATP) thus in the molecular balance of lay up period.The specific embodiments of the present composition provides the composition that can keep RBC suspension pH, and wherein said composition is added in the suspension with about 6.4 to 7.4 pH value.In a more particular embodiment, said composition can keep the pH value of red blood cell (RBC) suspension, and wherein said composition is added in the suspension to the pH that is lower than about 7.2 with 7.0.In a more particular embodiment, said composition can keep the pH value of red blood cell (RBC) suspension, and wherein said composition is to be added in the suspension to the pH less than 7.2 greater than about 7.1.

The present inventor has prepared and has been substantially free of muriatic interpolation liquid composite, and it produces negative effects to system astoundingly and allows the extra amount of buffer system that increases so that extra pH buffering to be provided.One embodiment of the invention are at being used at about 1 Aquo-composition to about 6 ℃ of following storage of red blood cells.This composition comprises: adenine; Dextrose; At least one nonmetabolizable film protectant sugar and pH buffer system.This pH buffer system contains the combination that can accept buffer on the physiology, and wherein buffer comprises the reagent that at least one can provide bicarbonate anion, and at least one can provide the reagent of phosphate radical anion and at least one that the reagent of sodium cation can be provided.Wherein, described pH buffer system exists with the amount that the pH of red blood cell (RBC) suspension that is enough to make above-mentioned composition to be added maintains following level, described level is enough at lay up period, in described red blood cell, set up and keep a kind of inhibition 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and be conducive to glucolytic molecular balance produces adenosine triphosphate (ATP) net profit thus in the molecular balance of lay up period.Said composition is substantially free of ectogenic chlorion." be substantially free of ectogenic chlorion " as used herein definition be though whether chlorion is provided, all do not have chlorion to be added in the composition.

Other embodiments are at the red cell suspension that contains any present composition, and wherein suspension is suitable for directly being infused into embodiment among the patient who needs this infusion.

Other embodiment of the present composition provides, and at least one can provide the reagent of sodium cation, and it is selected from sodium bicarbonate, sodium hydrogen phosphate and combination thereof.In a more particular embodiment, can provide at least a reagent of bicarbonate anion is sodium bicarbonate.Other embodiments provide at least a reagent that phosphate radical anion can be provided, and it is selected from sodium phosphate, sodium hydrogen phosphate, sodium phosphate and combination thereof.In a more particular embodiment, can provide at least a reagent of phosphate radical anion is sodium hydrogen phosphate.In other embodiment of the present composition, the combination that can accept buffer on the physiology also contains at least one can be provided on the physiology and can accept cationic reagent, and wherein cation is selected from H ⁺, potassium, ammonium, magnesium and combination thereof.

In another embodiment of the present composition, the film protectant sugar of at least one non-metabolism is mannitol.Some sugar alcohols particularly derived from the sugar alcohol (for example sorbierite, mannitol, xylitol, erythritol) of monose, are the hydrophilic small molecules that can easily spread some lipid barriers and may play a significant role in cell stability.Mannitol particularly, it is known in vivo as the antioxidant of hydroxyl scavenger.It has been brought into play main effect and has been considered to film protectant sugar in keeping cell membrane stability.Other little polyalcohol also can be used as film protectant sugar.It should be noted that glucose and mannitol have identical fractional dose, i.e. the 180g/ mole.Sugar alcohol can be by erythrocyte metabolism.

So-called Morie osmolarity is empirical numerical value as used herein.Morie osmolarity be pass complete semipermeable membrane (allow water from trip by and block a kind of film of solute transhipment fully) the measured value compared with pure water of the osmotic pressure that produces of solution.Morie osmolarity depends on the population in the solution but does not rely on the character of particle.The Morie osmolarity of simple solution equals the population that molar concentration multiply by per molecule.True solution may be more complicated.Protein with many equivalent/L may only have the very contribution of a small amount of to Morie osmolarity, because they are made up of minority very large " particle ".Not every ion all dissociates in solution.Cation may with other anion or combined with protein.Not every solution capacity all is moisture.Say truely and accurately, in all of these factors taken together all should be calculated in.Tension force with the Morie osmolarity height correlation and to describing the more useful value of biological cell environment, is the measured value that can compare with blood plasma by the osmotic pressure of cell membrane material.Morie osmolarity has been weighed the effective gradient of water, supposes all not infiltrations fully of all osmotic solutes.The population of calculating dissolving is very simple.For example, the NaCl solution of the glucose solution of 300mM and 150mM has identical Morie osmolarity.Yet it but is very different placing its behavior of cell of any of these solution.Tension force is the functional term of describing solution opposing ICV expansion trend.

Other embodiment provides has about 200 present compositions to about 310mOsm Morie osmolarity.In a more particular embodiment, composition has about 221 to about 280mOsm Morie osmolarity.In unusual specific embodiment, Morie osmolarity is about 270mOsm.

As described, RBCs is ATP with glucose (d-glucose=" dextrose ") metabolism.Refuse is lactate and proton.Proton is piled up, and has forced down pH and has suppressed further metabolism.Bicarbonate has been proposed as buffer system, and wherein it can be combined with proton, is converted into water and carbonic acid gas under the situation that has the RBC carbonic anhydrase.In the hold-up vessel that allows the carbonic acid gas diffusion, reverse reaction has been suppressed, and this reaction is towards generating CO ₂Direction carry out.Buffer system based on bicarbonate has had appreciable capacity.The bicarbonate that adds physiological concentration in the solution can produce pCO in solution ₂, it orders about the CO that spreads weekly up to 1 to 2mmol from 600mL PVC bag ₂Yet, synthetic and prolong effective storage period with regard to increasing ATP, before attempt RBC that preparation contains bicarbonate and store and add solution and fail.For example, Beutler (BAG-PM) record joins bicarbonate in the RBC stock solution, but failing to control higher pH causes ATP to be exhausted rapidly.

Because finding salt solution is not the necessary composition that RBC adds liquid composite, and the concentration of dextrose can be lowered and do not have negative effect to ATP is synthetic, so the present inventor can utilize the enhancing " effect " that produces in the solution parameter to go to improve and adjustment pH buffer system.Buffer system disclosed by the invention not only provides initial appropriate pH to adding liquid composite, and can also give the RBC suspension pH, and successively pH in the born of the same parents of RBC is adjusted to the value that can maximize synthetic ATP.Buffer system has realized that in storage period these pH regulate target.Therefore, the cushion effect of pH buffer system or intensity can be controlled wittingly.It is about 8 to about 9 composition that an embodiment of the present composition provides pH.In a more particular embodiment, pH is about 8.2 to about 8.8.In a more particular embodiment, the pH of composition is about 8.4 to about 8.6, and in unusual specific embodiment, the pH of composition is about 8.5.Another embodiment at be such present composition, wherein buffer system has such cushion effect in red blood cell (RBC), composition is added into the back, and to bring up at least about 2mEq and pH in the storage period in 6 weeks be 6.5 to 7.2.Buffer system disclosed by the invention should provide the cushion effect that is at least this value, also should be able to provide better cushion effect that further prolonged storage period to the RBC suspension.

The present inventor determined composition must composition scope, wherein the advantage of composition is open subsequently.In an embodiment of the present composition; composition comprises the adenine of about 1-3mM; about 20 to about 115mM dextrose, but about 15 to about 60mM non-metabolism film protectant sugar, about 20 to about 130mM sodium bicarbonate and about 4 arrives the sodium hydrogen phosphate of about 20mM.In a more particular embodiment, composition comprises the adenine of about 2mM, about 60 to about 100mM dextrose, but about 40 to about 60mM non-metabolism film protectant sugar, and about 22 to about 40mM sodium bicarbonate and about 7 arrives the sodium hydrogen phosphate of about 15mM.In a more particular embodiment, composition comprises the adenine of about 2mM, the dextrose of about 80mM, but the non-metabolism film protectant sugar of about 55mM, and the sodium bicarbonate of about 26mM and the sodium hydrogen phosphate of about 12mM, the pH of composition is about 8.5.

The present invention also provides the method embodiment.In a this embodiment, provide preservation red blood cell (RBCs) method through one period storage period.This method comprises: the whole blood sample through collecting that (a) will comprise the RBCs that remains to be stored and blood plasma mixes with anticoagulant solution, forms the suspension of collected whole blood thus; (b) the whole blood suspension of collecting is handled to remove blood plasma and concentrated RBCs through row, form the RBCs that concentrates thus; (c) RBCs that concentrates and a certain amount of Aquo-composition are mixed to form have about 35% suspension that arrives the RBC of about 70% volume; (d) cooling RBCs suspension is extremely about 1 ℃ to 6 ℃, and (e) stores the RBCs suspension that step is stored cooling according to standard.Aquo-composition mainly is made up of following material: adenine; Dextrose; But at least a non-metabolism film protectant sugar and pH buffer system.This pH buffer system contains the combination that can accept buffer on the physiology, it comprises the reagent that at least one can provide bicarbonate anion, at least one can provide the reagent of phosphate radical anion, can provide the reagent of sodium cation with at least one, wherein, described pH buffer system exists with the amount that the pH of red blood cell (RBC) suspension that is enough to make above-mentioned composition to be added maintains following level, described level is enough at lay up period, in described red blood cell, set up and keep a kind of inhibition 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and be conducive to glucolytic molecular balance produces adenosine triphosphate (ATP) net profit thus in the molecular balance of lay up period.This solution is just separated in order to dextrose and phosphate and bicarbonate are separated in the preparation.

RBCs used in this invention has separated from blood plasma in the normal processes of preparation component and resuspended RBCs in anticoagulant solution.In brief, will comprise exactly RBCs and blood plasma the standard whole blood sample (450.+/-.45ml) mix to form the whole blood suspension with anticoagulant solution (about 63ml).Also can increase or reduce liquor capacity pro rata to reflect the volume of different donor blood, for example 400.+/-.40ml-500.+/-.50ml.Then that the whole blood suspension is centrifugal in order to separate RBCs from blood plasma, form the RBCs that concentrates thus.The carrying out of whole process can use routine techniques to improve by reducing leukocyte.

Suitable anticoagulant comprises the conventional anticoagulant that becomes known for storing RBCs.Preferred anticoagulant comprises that pH is 5.5 to 8.0 citrate anticoagulation agent, CPD for example, the CPD of half strength etc.Most preferred anticoagulant is CPD.

Usually use specimen bag or PVC transhipment packing that the RBC suspension is stored in polyvinyl chloride (PVC) the storage blood bag of standard then, the size of wherein transhipment packing is with being changed by the volume of storage part.According to the step that the clinical practice of blood transfusion is put down in writing, editor Petz﹠amp; Swisher, Churchill-Livingston publishers, N.Y., 1981, the RBC suspension is stored under about 1 ℃ to 6 ℃.The All Files of quoting in context in this mode by reference is incorporated herein.In a specific embodiments of the inventive method, the suspension of RBCs is suitable for directly being infused in the patient's body that needs this infusion.And PVC storage blood bag is the standard storage blood bag of industrial approval; The present invention expects can use the numerous different sack that is suitable for storing the RBC suspension, for example comprises required suitable plastisizers.Relevant with sack or relevant with the container component of RBC storing technology composition is not discussed at this, but those skilled in the art can use many container technique to realize the present invention at an easy rate.

Interpolation solution of the present invention can also be used for making the RBC of freeze-drying rehydrated, or for example RBC is rehydrated with the freezing blood of the storage in fusing or blood component.

Preserve in the specific embodiments of RBCs method in the present invention, but at least a non-metabolism film protectant sugar is the monose from sugar alcohol that in a more particular embodiment, but non-metabolism film protectant sugar is mannitol.In other method embodiment, can provide at least a reagent of sodium cation to be selected from sodium bicarbonate, sodium hydrogen phosphate and combination thereof.In specific embodiment, it is sodium bicarbonate that at least a reagent of bicarbonate anion can be provided.Further embodiment is the inventive method at protection RBCs; wherein can provide at least a reagent of phosphate radical anion to be selected from sodium phosphate, sodium hydrogen phosphate, sodium phosphate and combination thereof; in a more particular embodiment, can provide at least a reagent of phosphate radical anion is sodium hydrogen phosphate.In other embodiment of the inventive method, the combination that can accept buffer on the physiology contains and comprises at least one and can provide on the physiology and can accept cationic reagent, and wherein cation is selected from H ⁺, potassium, ammonium, magnesium and combination thereof.

Further embodiment at be the method that the present invention preserves RBCs, wherein the Morie osmolarity of composition is about 200 to about 310mOsm.In specific embodiment, Morie osmolarity is about 221 to about 280mOsm, and in unusual specific embodiment, Morie osmolarity is about 270mOsm.Method of the present invention is provided in another embodiment, and wherein the pH of composition is about 8 to about 9.In specific embodiment, pH is about 8.2 to about 8.8, and in a more particular embodiment, the pH of composition is about 8.4 to about 8.6.In unusual specific embodiment, the pH of composition is about 8.5.Another embodiment that the present invention preserves the RBCs method provides, and buffer system has such buffer capacity in red blood cell (RBC) suspension, wherein bring up to 2mEq in the storage in 6 weeks after adding composition, and pH is between 6.5 to 7.2.

The present invention also provides the inventive method of preserving RBCs, and wherein, the pH that described composition operationally keeps having added red blood cell (RBC) suspension of described composition is about 6.4 to about 7.4.In concrete method embodiment, composition can keep the pH of red blood cell (RBC) suspension, wherein composition is about 7.0 to being added in the suspension less than 7.2 o'clock approximately at pH, in method embodiment more specifically, composition can keep the pH of red blood cell (RBC) suspension, wherein composition at pH for greater than about 7.1 and be added in the suspension less than 7.2 o'clock.

The method according to this invention at the necessary concrete scope of component of composition also is provided.In a method embodiment, composition comprises the adenine of about 1-3mM, about 20 to about 115mM dextrose, but about 15 to about 60mM non-metabolism film protectant sugar, and about 20 to about 130mM sodium bicarbonate and about 4 arrives the sodium hydrogen phosphate of about 20mM.In a more particular embodiment; composition comprises the adenine of about 2mM; about 60 to about 100mM dextrose; but about 40 to about 60mM non-metabolism film protectant sugar; about 22 to about 40mM sodium bicarbonate and about 7 arrives the sodium hydrogen phosphate of about 15mM; in unusual specific embodiment; composition comprises the adenine of about 2mM; the dextrose of about 80mM; but the non-metabolism film protectant sugar of about 55mM; the sodium bicarbonate of about 26mM and the sodium hydrogen phosphate of about 12mM, further, wherein the pH of composition is about 8.5.

In the method according to the invention, join in the concentrated RBC suspension adding solution, its amount is for being enough to provide restored concerning the RBCs for the treatment of effective dose in cell suspension.Preferably, the addition of adding solution arrives about 400mg for about 60ml, and preferred about 100 to about 150ml, most preferably from about 110ml.The consumption that this solution is general is to be 1: 4.5 (450mL collects whole blood to 100mL, and 500mL collects whole blood to 111mL, or equivalent) with the volume ratio of collecting whole blood.In the specific embodiments of the inventive method of protecting RBCs, composition is about 1: 4.5 with the volume ratio of collecting whole blood.In a more particular embodiment, the volume of composition is about 110mL, and the volume of collecting whole blood is about 500mL.

The volume fraction of RBC in the cell suspension after adding interpolation solution, is about 27 to 70% of total suspension namely.More preferably, the volume fraction of RBC is about 35 to about 50% in the cell suspension.Most preferably, the volume fraction of RBC is about 43% of total suspension in the cell suspension.

At lay up period, the present inventor's monitoring has also been collected and the healthy relevant data of red blood cell.As illustrated in fig. 1 and 2, the storage of carrying out according to the present invention makes red blood cell has better quality under low capacity and in long storage period.As mentioned above, it is apoptotic event that blood is stored " damage ", and erythrocyte membrane has experienced physiology and form changes, and this is equivalent to apoptosis.At lay up period, the surface area of known erythrocyte membrane is descending, and its shape just becomes double concave thus, and the surface area maximum that this can make under every volume spreads gas and nutrient easily, to the shape that is ball-type more, this is a kind of dead feature.Erythrocyte membrane is soft and deformable at first, is easy to by little capillary.Whole change of shape is accompanied by the film of extruding microvesicle (microvesicles), form spicula (spicules) at the red blood cell outer surface, thus, according to observations, cell is at the hedgehog that just is similar to thorn-like latter stage of this process (so this process refer to the echinocytosis variation, the net shape before cytolysis is called as acanthocyte).Thing followed fragility finally causes cytolysis and death.Determine that haemolysis speed can be as the index of this scope of activities and severity.Except causing at lay up period the unacceptable hemolysis levels, these metamorphosis are all pouring into by the acceptor patient internal trigger of storage of red blood cells purge mechanism again, and this has reduced the recovery behind the infusion and has reduced infusion efficient.Use makes osmotic fragility reduce and the rate of dissolution reduction according to composition of the present invention and the erythrocytic method of preservation of having used said composition.This has correspondingly improved surface of cell membrane long-pending confining force and form situation, and has therefore improved active erythrocytic recovery and reduced the removing in the acceptor patient body of the red blood cell that poured into again behind the infusion.

One embodiment of the invention provide the method for improving confining force and the inhibition RBC apoptosis of red blood cell (RBC) film at lay up period.This method is included in lay up period and stores RBCs in suspension, has wherein added composition of the present invention in the suspension.In specific embodiment, the concentration of microvesicle is compared with store the viewed concentration of red blood cell of identical time in containing the RBC suspension of AS-3, has reduced about 75%.

Another embodiment of the invention provides at lay up period and has reduced red blood cell (RBC) fragility and suppress the method for RBC haemolysis.This method is included in lay up period and stores RBCs in suspension, has wherein added composition of the present invention in the suspension.Another embodiment at be after storage and be infused in the patient's body that needs this infusion after can improve the viablity of red blood cell (RBCs), and reduce the method for clearance rate behind the patient RBCs infusion.This method is included in lay up period and stores RBCs in suspension, has wherein added composition of the present invention in the suspension.

Interpolation liquid composite of the present invention has been compared several advantages with the interpolation solution of prior art.The red blood cell that is stored in wherein can be stored the longer time, is at least for 8 weeks, compares with the solution of present permission, and the RBC of better radiation chromium-mark is arranged in restoring in 24 hours in vivo.Use composition of the present invention can reduce scope and the severity of storing damage significantly.At lay up period, acceptable haemolysis scope has appearred in red blood cell: being 0.2% the time in 6 weeks, is 0.4% when 8 weeks, storage latter stage of permission all at below 1% of FDA restriction.Be stored in the red blood cell that the present invention adds in the solution and show still less film loss at lay up period, reduce and the osmotic fragility reduction proves as the concentration of film microvesicle.Lay up period to the protection of film expection can help to be stored in the solution RBC so that its with lose more the cell of multimembrane and compare and have better flowability.Keep the physiological of normal film also to suppress to be present in the interior mechanism of acceptor patient body, this mechanism has triggered selective clearing and the erythrocytic destruction in the circulation.Therefore, the red blood cell of perfusion again among the acceptor patient just can be kept the longer time, and has improved and poured into erythrocytic long-term recovery again.In addition, the erythrocytic time-to-live of infusion improves the needs that should be able to reduce the repetition infusion and reduced associated danger thus concerning the patient.Solution of the present invention can also allow to have higher RBC ATP concentration, this expection that RBC ATP is secreted better late period and therefore improve its flowability and viability after cell is by infusion storing.In fact, solution of the present invention can be used when working with conventional gathering system, and wherein whole blood is collected generating fresh FP and donor blood platelet at random by this system in CPD or CP2D, or collects fresh whole blood at the critical moment.

The embodiment that below provides only is used for illustration purpose, and it should not be interpreted as limiting the scope of the present invention, and scope such as claim are determined.

Embodiment

Table 1

The interpolation liquid composite that is tried

?	AS-3	EAS-61	EAS-76v6	EAS-81
					NaCL	70	26	30	?
NaHCO ₃	?	?	30	26
					NaH ₂PO ₄	23	?	?	?
Na ₂HPO ₄	?	12	9	12
					Adenine	2	2	2	2
Sodium citrate	18	?	?	?
					Dextrose	55	110	50	80
Mannose	?	55	30	55
					Volume	110	170	170	110
pH	5.8	8.3	8.4	8.5

Embodiment 1

This embodiment has illustrated that the present invention adds performance characteristic and the advantage of an embodiment of liquid composite, and wherein composition is called EAS-81 (seeing Table 1).EAS-81 and Comparative Example A An S-3 (Nutricel, Pal Biomedical) are convention amount, and comparing embodiment EAS-61 and EAS-76v6 are so that dilution, bigger volume are provided more.EAS-81 and EAS-76v6 comprise bicarbonate.Referring to Fig. 1, the composition that wherein comprises bicarbonate is represented with circle, and is not had representing with rhombus of bicarbonate.The composition that volume is bigger is represented with solid figure, and the composition of conventional volume is represented with hollow figure.All volumes of interpolation liquid composite disclosed herein all are understood that the volume with respect to every 500mL unit whole blood, and its volume ratio is about 1: 4.5.

Carry out first embodiment as mixing (Pooling) research, in order to estimate the integrality the composition of stock solution stored for 10 weeks to the effect of RBC metabolism with in the PVC bag after.Mix (Pooling) and greatly reduce the otherness that conventional blood is stored research, just reduced the genetic diversity from different donor RBCs.Mixing is some cells of each donor to be placed some in each seminar keep conventional cell size simultaneously.In indirect antiglobulin test (IAT), do not have active RBC unit, be divided into 4 unit that ABO-is identical.Merge each group then, mixing and five equilibrium are to form four identical merge cellses.A unit during each mixes is used for of four researchs.

Composition AS-3 and EASs are disclosed in the table 1.By USP adenine, sugar and Sigma Chemicals, make by the salt that St.Louis, MO provide in the laboratory for EASs, and be aseptically filled in one liter of storage bag (Code4R2032, Baxter Healthcare, Deerfield, IL)." phosphate, pH and AS volume are to being stored in the effect of the RBCs in salt solution-adenine-glucose-mannitol solution referring to Hess etc." Transfusion, vol.40:1000-1006, in August, 2000, detailed description like that, at this by with reference to all being incorporated herein.Storage bag kept for two weeks down at 37 ℃.Then this solution is cultivated, and culture was hatched for two weeks again.Aseptic by not having to determine (SeptiCheck over bacterium and conk 7-14 days, Becton-Dickinson Microbiology Systems, Sparks, MD), and with solution with (Code4R2023 in the 600mLPVC bags that are placed in such as weight, Baxter Healthcare Corp., Deerfield, IL).Aseptic jockey is all used in all connections, and (SCD312, Terumo Medical Corp.Elkton MD) carries out.

The preparation of RBC unit:

(500 ± 55mL) use three-bag (triple-bag) gathering system, and (East Hills NY) collects 70mL CP2D solution for Item code#127-23, Pall Corporation in the blood unit of standard.The unit carries out leucocyte reduction (leukoreduced) with complete leucocyte reduction (leukoreduction) filter.The RBCs that concentrates makes by removing almost 65mL blood plasma in centrifugal 5 minutes then, afterwards, sterilely adds AS or the EAS of listed volume.The unit is stored in 1-6 ℃ of following 10 weeks, mixes weekly approximately and remove the 15mL sample.

External test

Leucocyte reduction (removing leucocyte-removing from whole blood products) is confirmed by flow cytometry.(Allentown PA) measures the concentration of total ferroheme (Hb) for Hematology Cell Counter System Series9110+, Baker by the clinical blood analyzer.MCV (MCV is determined by the microhematocrit of RBC counting and storage suspension).Hb in the supernatant uses the Drabkin determination method of improvement by spectrophotometric determination, " is used for measuring 5 slight improvements that the Drabkin ferrohemes of blood plasma ferroheme are measured in the 2000mg/dl scope as Moore etc.It is such that Biochem Med26:167-173 (1981) discusses, and all introduces by reference at this.Haemolysis percentage is determined by measuring free ratio and hematocrit value with total Hb.

RBC ATP concentration taking off among the albumen PRBCs in supernatant measured.The cell five equilibrium is mixed to precipitate hemalbumin with cold 10% trichloroacetic acid, under 2700x g centrifugal 10 minutes, and it is freezing until the experiment beginning down at-80 ℃ not have the albumen supernatant.ATP uses commercially available kit, and (St.Louis MO) passes through enzymatic assays for Procedures366-UV, Sigma Diagnostics.

Vim and vigour, bicarbonate and pH are blood gasanalyzer (Corning855, Ithica, NY) middle mensuration or calculating.Like this, pH measures and just carries out under 37 ℃.Extracellular sodium, potassium, chlorine, phosphate, lactate and glucose are chemical analyzer (Hitachi902Analyzer, Boehringer Mannheim Corporation, Indianapolis, IN) middle mensuration of sequencing.The average degree that the shape of RBC is changed to acanthocyte to spherocyte by discal cell is according to Usry, Moore, the method of and Manolo is carried out, this method is recorded in " Morphology of stored; rejuvenated human erythrocytes " among the Vox Sang28:176-183 (1975), introduces by reference in its entirety at this.Microvesicle is measured in the supernatant of storing latter stage.Blood plasma is through super centrifugal, and the vesicle precipitation is used Bradford method (BioRad, Richmond, CA) quantitative Tot Prot then with salt water washing three times.

The comparative test of 4 kinds of RBC stock solution the results are shown among Fig. 1 and 2.In all stock solution, RBC ATP concentration has raising (Figure 1A) in first week of storing.In EASs, ATP concentration continued to raise in second week, and kept than AS-3 height in during studying always.When storing for eight weeks and surpassing for eight weeks, the concentration of ATP is identical with the concentration of the EAS-61 of volume increase among the EAS-81, and EAS-78, it has volume and the bicarbonate buffer system of increase, and the concentration of its ATP is high slightly.

At lay up period, the haemolysis in all stock solution (erythrocytic destruction) all increase (Fig. 1 b).Yet the haemolysis among the AS-3 is higher.Reduction is arranged among the EAS-81 slightly, further reduction is arranged in other EASs.In containing the high volume solution of bicarbonate, haemolysis is extra the reduction not.As expected all be the hematocrit of the more low storage of AS volume and RBCs loss volume just more big (Fig. 1 c) at all solution of lay up period.

In comprising the EASs of bicarbonate lactate concentration at all time points all than higher (Fig. 1 d).Storing the Lactated gross yield in 8 week backs is 9mM in AS-3, is 12mM in EAS-61, is 13mM in EAS-81, is 15mM in EAS-78.The inside and outside pH of cell at all time points also all than higher, but the difference of internal pH does not reach statistical significance (Fig. 1 e and 1f).The time period that provides the bicarbonate loss that cushions in the suspension and PCO2 to increase and reduce has been provided for Fig. 1 g and 1h.

EAS-81 of the present invention and AS-3 store cell relatively carry out relatively disclosed the former the high ATP concentration of gained had the better energy utilization, haemolysis still less and better form, microvesicleization still less and small pH raise.Existing EAS ' s, especially EAS-76v6, in some aspects in addition performance better, but their bigger volumes cause having been produced bigger dilution to storing RBC, this can cause the hemodilution of multiple blood transfusion patient acceptor.

Embodiment 2

This embodiment has illustrated that EAS preparation of the present invention can store RBC8 week in the interpolation solution of conventional volume, it has better recovery, the film protectiveness of lower hemolytic and enhancing during 6 known all storage schemes.

12 volunteers that meet U.S. food and drug administration (FDA) and U.S. associating blood bank donor standard have been selected.The experimenter has donated 500mL (unite) whole blood, and it is collected in, and (East Hills NY), and is the leucocyte reduction for Item code#127-23, Pall Corporation, and it has almost removed about 65mL blood plasma in CP2D elementary (primary) bag.Add EAS-81,110mL, and the RBC solution that concentrates uprightly is stored under 1-6 ℃, wherein a half-sample is stored 6 weeks (n=6), and half stores 8 weeks (n=6).Storing the last week that finishes, the unit is carried out sterile sampling and cultivation.If cultivate and do not show growth, so just with 51-Cr a small amount of aliquot is carried out mark, and use Moroff method (protocol) that it is turned back in the donor to be used for the single label measurement that RBC restores.

Generate average and standard error figure, other descriptive statistics of storage group uses table procedure software, and (Redmond WA) calculates for Excel, Microsoft.Lactated output is calculated by beginning and end level in each storage system, and the volume of storage system is regulated with ferroheme and serum albumin content.(SPSS Inc., Chicago IL) generate the rectangular figure of RBC disengaging value (box plots) with SyStat Ver.6.

Be stored in its interior 87 ± 2% (Fig. 2) of recovery of bodies from body restored for 85 ± 5%, 8 weeks in 24 hours in the body after of RBC of the leucocyte reduction in 6 weeks among the CP2D/EAS-81.This long-time obviously unusual high return rate may be research small scale and the big reason of Different Individual blood group storage property difference.Yet these recoveries all generally are better than licensed stock solution, and can meet the permission (being to show greater than 75% recovery and haemolysis less than 1% for the U.S., is less than 0.8% greater than 75% recovery and haemolysis for Europe perhaps) of US and European.RBC haemolysis part is 0.2 ± 0.2 in the experiment of six weeks in this research, is 0.4 ± 0.2 in the experiment of 8 weeks.RBC microvesicle protein concentration is 8 ± 4mg/dL the time in 6 weeks, is 12 ± 6mg/dL when 8 weeks, and this is because only have an appointment 5% RBC ferroheme loss.

EAS-81 of the present invention has following several foundation in the good shelf characteric of storing the duration and improve aspect the infusion product physiological function.First, new pH regulating system can produce the buffering that continues at the experiment lay up period to suspension solution, this is enough to pH with the intracellular region territory and remains on and be enough to promote born of the same parents' inner equilibrium and carry out and consume in the scope that direction carries out to produce ATP away from 2,3-DPG to the glycolysis direction.In addition, in this preparation the outer phosphate concn height of born of the same parents owing to the outer Ca of born of the same parents ⁺⁺The restriction of concentration, this has suppressed several apoptotic processes for example phosphatide confusion (scrambling) and film distortion and loss successively.This causes by the recovery of infusion RBCs behind infusion and makes moderate progress, and improving the performance of film, purge mechanism brought out fast and causes and recoverablely in time expand beyond the infusion phase improved by infusion RBCs percentage in this had further reduced in by the infusion patient body.The EAS-81 preparation, it does not contain the Morie osmolarity that NaCl keeps suiting to help to improve the quantity of specific buffer compounds, and it has better effect, to adding RBC solution suspension longer pH modulability is arranged, and more phosphate, it can make and improve Ca ⁺⁺Concentration and the concentration dependent unfavorable cell event minimization of reduction ATP.In addition, the preparation EAS-81 of conventional volume be particularly suitable for being used in repeatedly and the patient of a large amount of infusions in.

Claims

1. at about 1 ℃ of Aquo-composition to about 6 ℃ of following storage of red blood cells, said composition mainly is made up of following material:

Adenine;

Dextrose;

But at least a non-metabolism film protectant sugar and

The pH buffer system,

Wherein the pH buffer system contains the combination of physiologically acceptable buffer, wherein buffer comprises at least a reagent that bicarbonate anion can be provided, at least a reagent and at least a reagent that sodium cation can be provided of phosphate radical anion can be provided, wherein, described pH buffer system exists with the amount that the pH of red blood cell (RBC) suspension that is enough to make above-mentioned composition to be added maintains following level, described level is enough at lay up period, in described red blood cell, set up and keep a kind of inhibition 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and be conducive to glucolytic molecular balance produces adenosine triphosphate (ATP) net profit thus in the described molecular balance of lay up period.

2. at about 1 Aquo-composition to about 6 ℃ of following storage of red blood cells, said composition comprises:

Adenine;

Dextrose;

But at least a non-metabolism film protectant sugar and

The pH buffer system,

Wherein the pH buffer system contains the combination of physiologically acceptable buffer, wherein buffer comprises at least a reagent that bicarbonate anion can be provided, at least a reagent and at least a reagent that sodium cation can be provided of phosphate radical anion can be provided, wherein, described pH buffer system exists with the amount that the pH of red blood cell (RBC) suspension that is enough to make above-mentioned composition to be added maintains following level, described level is enough at lay up period, in described red blood cell, set up and keep a kind of inhibition 1,3-DPG synthetic 2,3-diphosphoglyceride (DPG) and be conducive to glucolytic molecular balance, in the described molecular balance of lay up period, produce adenosine triphosphate (ATP) net profit thus, wherein, described composition is substantially free of the chlorion of external source.

3. improve the method for confining force and the inhibition RBC apoptosis of red blood cell (RBC) film at lay up period, this method comprises: at lay up period RBCs is stored in the suspension that has added composition as claimed in claim 1.

4. reduce red blood cell (RBC) fragility and suppress the method for RBC haemolysis at lay up period, this method comprises: at lay up period RBCs is stored in the suspension that has added composition as claimed in claim 1.

5. improve the viablity of storing the back and being infused into back red blood cell (RBCs) in the patient's body that needs this infusion, the method of clearance rate behind the reduction patient RBCs infusion, this method comprises: at lay up period RBCs is stored in the suspension that has added composition as claimed in claim 1.

6. preserve red blood cell (RBCs) through the method for one section period of storage, it comprises:

(a) whole blood sample through collecting that will comprise the RBCs that remains to be stored and blood plasma mixes the suspension of the collected whole blood of formation with anticoagulant solution;

(b) the whole blood suspension of collecting is handled to remove blood plasma and concentrated RBCs through row, form the RBCs that concentrates thus;

(c) RBCs that concentrates is mixed with a certain amount of Aquo-composition, wherein this amount is enough to make RBCs suspension 27% RBCs that arrives about 70% volume that has an appointment;

(d) cooling RBCs suspension is extremely about 1 ℃ to 6 ℃, and

(e) store the RBCs suspension that step is stored cooling according to standard

Wherein Aquo-composition mainly is made up of following material:

Adenine;

Dextrose;

But at least a non-metabolism film protectant sugar and

The pH buffer system,

7. preserve red blood cell (RBCs) through the method for one section lay up period, it comprises:

(a) whole blood sample through collecting that will comprise the RBCs that remains to be stored and blood plasma mixes with anticoagulant solution, forms the suspension of collected whole blood thus;

(c) RBCs that concentrates is mixed with a certain amount of Aquo-composition, wherein this amount is enough to make the RBCs suspension to have the interior RBCs percent by volume of standard blood bank procedure prescribed limit;

(d) cooling RBCs suspension is extremely about 1 ℃ to 6 ℃, and

(e) store the RBCs suspension that step is stored cooling according to standard

Wherein Aquo-composition mainly is made up of following material:

Adenine;

Dextrose;

But at least a non-metabolism film protectant sugar and

The pH buffer system,

8. preserve red blood cell (RBCs) through the method for one section period of storage, it comprises:

(b) the whole blood suspension of collecting is handled to remove blood plasma and concentrated RBCs, form the RBCs that concentrates thus;

(c) RBCs that concentrates is mixed to the Aquo-composition of about 400mL with about 60mL;

(d) cooling RBCs suspension is extremely about 1 ℃ to 6 ℃, and

(e) store the RBCs suspension that step is stored cooling according to standard

Wherein Aquo-composition mainly is made up of following material:

Adenine;

Dextrose;

But at least a non-metabolism film protectant sugar and

The pH buffer system,

9. contain the combination of claim 1 composition that is arranged in storage blood bag, wherein this storage blood bag has enough volumes in order to can comprise about 60ml to the interpolation solution and the whole blood of about 270ml to about 2250ml volume of about 400ml volume.

10. Morie osmolarity is the Aquo-composition of 270mOsm, and it was used in about 1 to about about 8 weeks of 6 ℃ of following storage of red blood cells, and wherein composition mainly is made up of following material:

The 2mM adenine;

The 80mM dextrose;

But at least a non-metabolism film protectant sugar of 55mM is mannitol for example; With

The pH buffer system,