the following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention . various modifications , however , will remain readily apparent to those skilled in the art , since the general principles of the present invention have been defined herein specifically to provide a simple procedure for enhanced production of factor viii from collected plasma . the traditional method for producing clotting factors , as well as many of the presently used methods , operate because many of the plasma proteins responsible for blood clotting precipitate ( i . e ., form cryoprecipitate ) from solution at low temperatures . when a protein solution is frozen , ice crystals form and protein molecules , which are excluded from the crystals become increasingly concentrated . cooling or freezing the water also lowers the chemical activity of the water . depending on the particular proteins , the proteins may actually fall out of solution , i . e ., form a precipitate , if the protein more readily interacts with itself or with other proteins than with water . when the chemical activity of water is lowered such precipitation is favored . such precipitation may denature the proteins ( make them irreversibly insoluble ), so it is usual to freeze protein solutions rapidly and to a low temperature ( i . e ., − 20 ° c . or lower ) to minimize the formation of ice crystals and to prevent the growth of those crystals that do form . this is done to limit protein denaturation on ice crystal surfaces . blood coagulation enzymes are extremely sensitive . even when freezing is carried out with great care , ice crystals may cause “ activation ” of the prothrombin complex , resulting in spontaneous clot formation and loss of coagulation factors to proteolysis and / or clot formation . it now appears that the most significant amount of such activation occurs during the thawing process as opposed to during the freezing process . the first step in the typical procedure for producing plasma cryoprecipitate is to centrifuge whole blood to separate the plasma from the red blood cells . this procedure is well known in the art and is often accomplished in special centrifuges that hold individual blood bags so that the plasma / red cell separation occurs without even opening the blood bag . following the centrifugation , it is common practice to express the supernatant plasma into a “ satellite ” blood bag for further processing . once the plasma is separated from red and white blood cells , the typical procedure is to rapidly freeze the plasma and to then slowly thaw the frozen plasma at about 4 ° c ., during which thawing the clotting factors and other proteins form a cryoprecipitate which can be readily harvested by filtration or centrifugation . this cryoprecipitate is not rendered irreversibly insoluble and can be readily redissolved in a saline buffer , or even water , as is well known in the art . cryoprecipitation is generally believed to result when the removal of water from the immediate vicinity of the protein molecules causes the protein molecules to preferentially associate with each other rather than with water . this “ removal ” of water may represent changes in the solubility of the proteins with changes in temperature ( i . e ., water becomes less effective at dissolving the proteins ). the process may also be accomplished or enhanced by using additives which “ tie up ” the water and cause it to interact with the proteins to a lesser degree . these additive substances can be any of a number of hydrophilic materials such as ethanol , polyethylene glycol , heparin , pluronic rtm polyol polymers and various “ salts ” such as ammonium sulfate or ammonium acetate . the “ salting out ” of proteins from solution is a classical biochemical procedure . these and other materials used to increase the yield of cryoprecipitate generally operate to decrease the effective activity of water in the mixture . that is , the water molecules preferentially interact with the added hydrophilic material instead of with the proteins . this permits the proteins to interact with each other and , therefore , precipitate from solution . similarly , lowering the temperature — especially to the freezing point — also decreases the activity of water , allowing protein - protein interactions to predominate . the hydrophilic additives just mentioned have the advantage of being relatively inexpensive and easy to use . however , their use usually necessitates additional washing steps to ensure that the additives are not carried over into the final product . some additives may also damage or denature the labile clotting factors one is seeking to purify . the present inventor has discovered that one of the agents frequently used as an anticoagulant in blood fractionation unexpectedly serves to enhance cryoprecipitate formation . citrate ( trisodium citrate or similar salts as well as derivatives of other low molecular weight carboxylic acids such as isocitric acid ) has unusually favorable properties when used in blood fractionation procedures at levels significantly higher than those traditionally used as an anticoagulant . citrate is a fairly effective chelator of calcium ions . by effectively lowering the calcium ion level , citrate inhibits a considerable variety of blood clotting pathways which depend on the presence of calcium ions . however , citrate has not been employed as an agent to simultaneously prevent loss through activation and to enhance the preparation of cryoprecipitate proteins from plasma . the following table shows the enhanced production of cryoprecipitate caused by increasing the level of trisodium citrate in plasma . as the citrate is increased , the weight of recovered cryoprecipitate is increase . when the cryoprecipitate is redissolved in a fixed quantity of buffer or water , the increasing amount of cryoprecipitate yields increasing amounts of factor viii and fibrinogen as compared to the original plasma . it seems reasonable to speculate that since one action of citrate is to inhibit the activation of clotting factors , which act as proteases when activated , inhibition of activation prevents digestion of clotting proteins thus increasing the yield of these proteins . it is important to appreciate that the increased amounts of citrate are added to the plasma as soon as practicable — preferably before any freezing of the plasma . if the plasma is frozen without the added citrate , it is imperative that added citrate be present during the thawing process so that losses due to activation of clotting factors during thawing are prevented . these results indicate that as the citrate concentration is increased the amount of recovered clotting factors increases linearly . there is a further increase of cryoprecipitate with 15 % citrate ; however , at that concentration of citrate it appears that there is an increase in the precipitation of other proteins . the optimum concentration lies between about 10 % and about 15 % weight / volume citrate . tests have shown that besides more than 95 % of the factor viii and fibrinogen , virtually all of the fibronectin and the von willdebrand &# 39 ; s factor become concentrated in the citrate - enhanced cryoprecipitate . further insight into the citrate effect is gleaned by analyzing the distribution of citrate in a typical cryoprecipitate experiment . for this experiment , one unit ( about 200 ml ) of plasma was brought to 10 % wt / vol . trisodium citrate . the citrate stock solution was adjusted to neutral ph with hcl or acetic acid prior to use , and in all experiments ph measurements showed that natural buffering of the plasma prevented significant changes in ph . this citrate - treated plasma was frozen and cryoprecipitate was collected in the usual manner . it important that the thawed material be gently mixed for sufficient time ( 12 hours ) to permit maximal precipitation to occur . as an aside , in producing citrate cryoprecipitate it is preferred to add the citrate prior to freezing , but good results are achieved by adding the citrate during the thawing process ( to the frozen plasma before thawing actually starts ). as will be demonstrated below , actual freezing is not necessary . the volume of cryoprecipitate formed from the unit of plasma was approximately 20 ml — that is , 10 % of the total volume . surprisingly , an analysis of the cryoprecipitate and the supernatant plasma showed that about 12 g ( 60 %) of the citrate was concentrated in the cryoprecipitate with only 40 % being left in the supernatant . this indicates that there is a strong interaction between the cryoprecipitate proteins and the citrate . the proteins become “ citrified ” or “ citrated ” upon incubation with elevated concentrations of citrate . further , while normal cryoprecipitate can be redissolved in water or buffer , citrated cryoprecipitate is somewhat less soluble in water . it is soluble , however , in saline buffer and most soluble when the buffer contains citrate . one way of explaining these phenomena is to assume that the multiple negative charges on the citrate molecule are interacting with positive charges on the cryoprecipitate proteins to cross - link them . added citrate “ satisfies ” these positive charges so that cross - linking is diminished . because of the inclusion of clotting proteins in the cryoprecipitate , it is tempting to theorize that the clotting proteins share some sort of positive charge motif that interacts with the citrate molecules . it may be that other proteins will also become “ citrified ” if incubated with a sufficiently high concentration of citrate . in summary , compared to “ normal ” cryoprecipitate citrated cryoprecipitate contains essentially all of the fibrinogen , fibronectin , factor viii and von willdebrand &# 39 ; s factor . the citrated cryoprecipitate may also contain other minor factors ( like factor xiii ) not yet assayed in these experiments . what may be important is what the citrated cryoprecipitate does not contain . as was mentioned above , it has been found that addition of citrate to frozen plasma during the unfreezing process appears to be almost as effective at increasing the amount of cryoprecipitate as adding the citrate prior to freezing . of course , in most cases it is more convenient to add the citrate to the blood bags prior to collection or expressing the plasma , or perhaps during the pooling of plasma prior to freezing . however , there are cases where pooled plasma is stored and shipped in the frozen state so that it is a significant advantage that the new enhanced citrate process can be used with such plasma even if the plasma was frozen before the new process was even invented . however , for best results the added citrate must be present during the thawing process . increasing the citrate concentration after thawing is not nearly as effective . in investigating this phenomenon it was discovered that freezing is not even necessary . in one experiment five 40 ml aliquots of human plasma were brought to 10 % wt / v trisodium citrate by the addition of 10 ml aliquots of a 50 % wt / v trisodium citrate stock solution . after mixing the aliquots were stored for 24 hours at 4 ° c . at the end of this time a large white precipitate had formed in each sample . the samples were centrifuged at 1 . 500 × g for 10 minutes in a refrigerated centrifuge to pellet the precipitate . the supernatant was carefully poured off , and each pellet was redissolved in 10 ml of 0 . 9 % nacl . a check of ph showed that it remained in the normal physiological range . calcium chloride was added to the solutions to overcome the citrate ( that is , to substitute for the calcium sequestered by the citrate so that clotting assays could proceed normally ), and each solution was sent to an independent laboratory for determination of factor viii and fibrinogen . the results are shown in the following table these results show that essentially all of the fibrinogen and factor viii ended up in the pellet . since the pellet from 40 ml of plasma was resuspended in 10 ml of saline one would expect a four - fold increase if all of these proteins were in the pellet . this is essentially what the tests show within their margin of error . similarly , the fibrinogen readings are about four times higher than normal . the small amount of factor viii and fibrinogen remaining in the supernatant is below the detection limits of the tests . this finding shows that it is possible to dispense with the cumbersome freezing and thawing steps altogether . with this method “ cryo ” takes on it &# 39 ; s preferred etymological meaning of “ icy cold ” rather than frozen . in fact , it appears that even icy cold is not strictly necessary . the following table shows the results of an experiment carried out exactly like the previous experiment except that the aliquots were allowed to rest for 24 hours at room temperature ( approximately 21 ° c .) prior to centrifugation . the results show that the separation was almost as good as at the lower temperature . further experimentation is necessary to determine whether 4 ° c . is a “ magic value ” or if some temperature lower than 21 ° c . but higher than 4 ° c . will produce optimum results . also , it is possible that a longer time at 21 ° c . will produce improved results . in any case , the difference between the results at 21 ° c . and 4 ° c . is small . either of these temperatures with citrate produces yields superior to current frozen cryoprecipitates without additional citrate . it would appear that simple incubation with elevated levels of citrate allows binding of the citrate or “ citrification ” of the proteins which results in precipitation . when the citrate level is reduced ( as in resuspension in saline ) the proteins readily go back into solution — indicating that they are not damaged by the “ citrifying ” process . the enhanced production of cryoprecipitate according to the present invention opens up the possibility of readily preparing a clotting factor concentrate from single donors or small pools of donors without any freezing step and without using chromatography . because the amount of clotting factor recoverable from a single unit of blood is generally large enough only for pediatric treatment , it is usually necessary or desirable to pool the plasma from a small , defined pool of donors ( usually fewer than ten donors ). by using a small and consistent donor pool , the possibility of blood - borne infection can be significantly decreased . to produce optimal single or limited donor pool clotting factor plasma is first collected to contain an optimal concentration of trisodium citrate . the optimum concentration is between 10 % and 15 % wt / vol . with about 12 % wt / vol . being a preferred concentration in many cases . one means of collecting the plasma is to centrifuge freshly collected units of whole blood in a blood bag centrifuge as is well known to those of skill in the art . at that point the supernatant plasma can be expressed into a separate blood bag containing sufficient stock citrate solution ( e . g ., 50 % wt / vol . trisodium citrate at ph 7 . 0 is convenient ) to bring the final citrate concentration to the desired level . if a pool is to be made , several units of plasma can be expressed into a single large blood bag . other means of achieving the same end will be apparent to those of ordinary skill in the art . for example , plasma collected by plasmapherisis can be collected directly into blood bags containing the extra citrate or the extra citrate can be added following collection . the preferred method is to store the citrated plasma in the cold ( 4 - 7 ° c .) for 24 hours . during this time a heavy cryoprecipitate will form ; after the cryoprecipitate has completely formed , it is separated from the supernatant plasma . again , centrifugation of the blood bag is a good method of achieving this separation of cryoprecipitate and supernatant plasma although filtration of other methods may be used . although cold precipitation is the preferred method , the plasma may also be frozen and the precipitation stage performed following thawing . the least preferred method is to freeze without added citrate and to add the citrate stock solution to the frozen plasma prior to thawing . the resulting cryoprecipitate or the components thereof can advantageously be used to treat congenital as well as “ acquired ” deficiencies . fore example , the material is useful in the treatment of hemophilia , liver disease , transplant cases and sepsis . as explained above , the clotting factors are essentially all present in the cryoprecipitate which can be redissolved in water or saline . however , merely redissolving the cryoprecipitate will produce a solution that is primarily fibrinogen . if sufficient amounts of this solution were administered to a patient to provide normal levels of clotting factors , the patient would receive a tremendous excess of protein mostly in the form of fibrinogen . therefore , some method must be used to decrease the amount of fibrinogen relative to the factor viii . it is known in the art that extracting the cryoprecipitate with cold saline preferentially dissolves the clotting factors while leaving most of the cryoprecipitate ( fibrinogen and fibronectin ) as a solid . for example , in one experiment cryoprecipitate was produced according to the above method using 12 % wt ./ vol . trisodium citrate . equal amounts of cryoprecipitate were resuspended for 30 minutes at 9 - 10 ° c . in a volume of cold buffer equal to the volume of cryoprecipitate . a typical unit of blood ( approximately 250 ml ) yields around 20 ml of cryoprecipitate . the average value of factor viii in the starting blood is 1 unit / ml so that the cryoprecipitate should contain between 200 and 300 units of factor viii activity . either cold 0 . 9 % saline ph 7 . 0 or cold 0 . 3m calcium chloride in 0 . 9 % saline ph7 . 0 was used as buffer for the extraction . following the extraction in the cold ( i . e ., below about 10 ° c . ), the material was recentrifuged to pellet the undissolved material . the supernatants were assayed , and it was discovered that the saline extract contained 4 . 1 units / ml of factor viii while the calcium saline extract contained 4 . 3 units / ml of factor viii . these are essentially equivalent amounts due to the level of precision of the assay . further , the amounts of factor viii extracted were nearly 100 % of that available . the most striking difference is seen when the amount of fibrinogen in the extract is measured . it is believed that the addition of calcium ions prevents the dissolution of fibrinogen . depending on the experiment the amount of fibrinogen in the calcium extraction varied from one half to less than one fifth as much fibrinogen ( generally in the range of 100 mg / dl ) as compared to traditional extraction methods . this level of fibrinogen is sufficiently low as to be almost negligible in terms of therapeutic administration . lowering the ph of the extraction medium to ph 5 . 5 may slightly lower the amount of factor viii extracted but further reduces the level of fibrinogen to essentially zero . thus , it is possible to readily produce a factor viii solution with levels of fibrinogen that are not significant when the solution is used to treat a patient . table i gives a clearer picture of the purification attained by the present invention . the table shows amounts of protein in mg and is adjusted so that each starting fraction ( e . g ., cryoprecipitate ) contains 100 units of factor viii activity . ahf stands for anti - hemophilia factor , a semi - purified factor viii concentrate . ip indicates intermediate purity while hp indicates high purity . note that traditional cryoprecipitate shows an apparent higher activity of factor viii . this is because the citrated cryoprecipitate of the present invention has relatively more fibrinogen than traditional cryoprecipitate ( that is , more total protein that is not factor viii ). as shown above when expressed on overall recovery of factor viii , citrated cryoprecipitate contains essentially all of the factor viii present in the original plasma whereas traditional cryoprecipitate does not . the other proteins present in the citrated cryoprecipitate ( albumin and α , β , and γ globulins ) are present primarily as trapped inclusions and are present in essentially the same proportions as in traditional cryoprecipitate . the improvements in purity when going from cryoprecipitate to ip - ahf to hp - ahf are occasioned by the removal of fibrinogen , albumin and globulins . it can be seen that the saline extract of citrated cryoprecipitate is nearly as pure as ip - ahf on a total protein basis . the inventive calcium extract is considerably better than ip - ahf but not as good as hp - ahp on a total protein basis . low ph calcium extraction , not shown in the table , yields a product even lower in total protein having essentially no fibrinogen . thus , when combined with citrated cryoprecipitate , the inventive extraction method allows simple production of a pure ahp concentrate from single donors or limited donor pools . ( values expressed as mg of protein giving 100 units of facto viii activity ) the inventive method of ahf production as diagrammed in fig1 consists of first producing citrated cryoprecipitate . preferably this is prepared from single donor plasma or from plasma pools produced from a limited number of donors . ideally , the required concentration of citrate ( 10 - 15 % wt ./ vol . trisodium citrate ) is added as soon as practicable after plasma collection . preferably , the citrated cryoprecipitate is collected after holding the plasma at about 4 - 6 ° c . for about 24 hours without freezing . it is also possible to freeze the plasma if it is not convenient to immediately effect separation of the citrated cryoprecipitate . in that case , the plasma is later thawed and held in the cold to allow complete formation of the cryoprecipitate . the cryoprecipitate is them separated from the supernatant by centrifugation or filtration . the supernatant plasma can be used for further fractionation or as a blood volume expander . the citrated cryoprecipitate is then extracted with the calcium saline extraction medium . this yields an ahp concentrate that can be used immediately in therapy . if desired calcium and or citrate can be removed from the concentrate using chromatographic and ultrafiltration methods well known to those of skill in the art . thus the present invention makes is possible for a blood bank to provide high quality ahp using simple equipment and procedures . the undissolved material following low temperature calcium extraction is primarily fibrinogen ( and fibronectin ). partly because of the added calcium it is possible to cause this material to gel if the temperature is raised to about 50 ° c . for about five minutes . at a lower temperature ( i . e ., room temperature ) the material will eventually gel but heating above room temperature greatly accelerates the process . most likely this is a clotting phenomenon mediated by one of the alternative coagulation pathways and potentiated by the added calcium ions . in one experiment the supernatant ( containing the concentrated clotting factors ) was removed and the fibrinogen pellet rinsed with cold buffer prior to the heating . in a second experiment the fibrinogen was heated without carefully removing the supernatant . in either case the material gelled forming a transparent semisolid which became increasingly opalescent and tough over the ensuing 12 hours . however , after 24 or so hours the material from the second experiment began to liquefy suggesting that the supernatant had contributed plasminogen which digested the fibrin . this provides a simple method for preparing fibrin / fibrinogen membrane or fabric . after the clotting factor concentrate is withdrawn ( e . g ., in a sterile blood bag ), it is possible to rinse the fibrinogen precipitate as necessary and mold it into a thin sheet all without opening the bag and compromising sterility . once the fibrinogen has been properly molded , the bag is heated to form the fibrin / fibrinogen fabric . depending on the desired strength of the material , it can be allowed to “ harden ” for eight or so hours prior to use . the fibrin / fibrinogen material can also be reinforced by embedding a mesh in the thin sheet . because one of the advantages of the fibrin material is that it is ultimately absorbed by the body , it is advantageous to make any reinforcing mesh from a biodegradable or absorbable material such as those commonly used to produce absorbable suture material . while the preferred method is ideal for use by hospitals to prepare fibrin fabric immediately prior to surgery ( possibly using autologous blood ), it is also possible to lyophilize the fibrin fabric so that it can be produced in advance and at remote locations . the invention covers the process and products obtained by the process . the following claims are thus to be understood to include what is specifically illustrated and described above , what can be obviously substituted and also what incorporates the essential idea of the invention . the illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention . therefore , it is to be understood that , within the scope of the appended claims , the invention may be practiced other than as specifically described herein .
the blood collection , processing and transfer by separation of discrete components containing additional citrate in one or other of collection or processing bag provides for enhanced yield and purity of cryoprecipitate . inhibiting the activation or denaturation of blood components including blood cells and plasma proteins and with the removal of the activated and denatured components thereby improving safety and efficacy of end products . the inventive process is particularly suited to an improved extraction process to yield concentrated clotting factors from single donors or limited pools without use of chromatography . following extraction the remaining cryoprecipitate can advantageously be formed into a fibrin fabric used in surgeries and in the treatment of wounds .