SAFE HUMAN TRANSFUSION BLOOD
Background of the Invention This invention relates to human transfusion blood, and derivatives thereof. Iodine was officially recognized by the Pharmacopeia of the United States in 5 1930, also as tincture iodine (tincture of iodine) and linimentum iodi (liniment of iodine). Clinicians and microbiologists described a great number of experimental data and clinical applications. Despite the successes that have been achieved with iodine, it was ascertained early that it also possesses properties imsuitable for practical application. 10 Although exact details about the killing of a living cell by the I2 molecule
(or one of the reaction products occurring in aqueous solution) are not known, it can be assumed that iodine reacts with basic N-H functions that are parts of some amino acids (e.g., lysine, histidine, arginine) and the bases of nucleotides (adenine, cytosine, and guanine) forming the corresponding N-iododerivatives. By this reaction, 15 important positions for hydrogen bonding are blocked, and a lethal disorder of the protein structure may occur. Iodine oxidizes the S-H group of the amino acid cysteine, through which the connections of protein chains by disulfide (-S-S-) bridges, as an important factor in the synthesis of proteins, are lost. Iodine reacts with the phenolic group of the amino acid tyrosine, forming mono- or diiodo-derivatives. In 20 this case, the bulk of the iodine atom(s) in the ortho position may cause a form of steric hindrance in the hydrogen bonding of the phenolic OH group and with the carbon-carbon double bond (C=C) of the unsaturated fatty acids. This could lead to a change in the physical properties of the lipids and membrane immobilization. Iodine - polymer complexes, e.g., with poly(vinylpyrrolidone) (PVP), and 25 complexes of iodine with nonionic surfactants, eg, polyethylene glycol ιnono(nonylphenyl) ether have been used with considerable success. However, use in direct contact with labile biological materials has been limited because either the ^ killing power of iodine is dissipated in the biological material or damages the biological material. ~~
'* 30 Iodine is capable, in certain circumstances, of killing all classes of pathogens encountered in nosocomial infections: gram-positive and gram-negative bacteria, mycobacteria, fungi, yeasts, viruses and protozoa. Most bacteria are killed within 15
to 30 seconds of contact. Iodine is generally considered to be an excellent, prompt, effective microbicide with a broad range of action that includes almost all of the important health-related microorganisms, such as enteric bacteria, enteric viruses, bacterial viruses, and protozoan cysts, if the sometimes severe limitations inherent in its use are overcome. Mycobacteria and the spores of bacilli and clostridia can also be killed by iodine. Furthermore, iodine also exhibits a fungicidal and trichomonacidal activity. As to be expected, varying amounts of iodine are necessary to achieve complete disinfection of the different classes or organisms. Within the same class, however, the published data on the disinfecting effect of iodine correspond only to a small extent. In particular, the published killing time of spores and viruses are widely disparate.
However, iodine is consumed by proteinaceous substrates and its efficacy as a disinfectant is reduced at certain antiseptic applications. This is due to a reducing effect of the material to be disinfected which leads to the conversion of iodine into non-bactericidal iodide. Thus, not only the reservoir of available iodine is diminished but also the equilibrium of triiodide is influenced as well. Both of these effects cause a decrease in the proportion of free molecular iodine, the actual anti-microbial agent. In whole blood, a strong decrease of the concentration of free molecular iodine occurs, while, in the presence of plasma, it remains practically unchanged. Durmaz, et al, Mikrobiyol. Bid. 22 (3), 1988 (abstract); Gottardi W, Hyg. Med. 12 (4). 1987.
150-154. Nutrient broth and plasma had little inactivating activity but 1 g hemoglobin inactivated 50 mg of free I; experiments with 12SI showed that uptake of I by [human] red cells occurred rapidly. Optimal antimicrobial effects in clinical use should be achieved in relatively blood-free situations. Povidone iodine produced a potent and sometimes persistent bactericidal effect towards bacteria on healthy skin.
Lacey, R. W '., J Appl Bacteriol 46 (3). 1979. 443-450. The bactericidal activity of dilute povidone-iodine solutions is inhibited to the greatest extent by blood, followed by pus, fat and glove powder. Zamora J L; Surgery (St Louis) 98 (1). 1985. 25-29; Zamora, Am. J. Surgery, 151, p. 400 (1986); see also, Waheed Sheikh, Current Therapeutic Research 40, No. 6, 1096 (1986). Van Den Broek, et al, Antimicrobial
Agents and Chemotherapy, 1982, 593-597, suggests that povidone-iodine is bound to cell wall proteins leaving little for interaction with microorganisms in the liquid phase
(See, also, Abdullah, et al, Arzneim.-Forsch./Drug Res. 31 (I), Nr. 5, 828).
Ninneman et al, J. of Immunol. 81, 1265 (1981) reported that povidone-iodine was absorbed in serum albumin and it is know that povidone-iodine is bound to albumin.
Iodine is used widely in human medicine is the disinfection of skin, (e.g., the preoperative preparations of the skin, the surgical disinfection of hands, the disinfection of the perineum prior to delivery, and the disinfection of the skin prior to infections and transfusions). Iodine preparations are also used for therapeutic purposes, e.g., the treatment of infected and burned skin but is a strong irritant.
Iodine has also been used for the disinfection of medical equipment, such as catgut, catheters, knife blades, ampules, plastic items, rubber goods, brushes, multiple-dose vials, and thermometers. The use of iodine as an aerial disinfectant has been advocated since 1926.
The use of "oxidizing iodine" including "compounds incorporating molecules of oxidizing iodine" e.g. absorbed or grafted on a purified vegetable carbon, as blood- contacting reagents having bactericidal and bacteriostatic action are mentioned in passing in connection with an autotransfuser device in U.S. Patent 4,898,572,
Surugue nee Lasnier, et al but without any explanation or elucidation. It may be inferred that Surugue used an iodine-treated charcoal, but no description is provided.
Various authors have tried to summarize the disinfecting properties of iodine and the other halogens by reviewing the literature and analyzing the existing data.
The most important conclusions are:
(1) A standard destruction (i.e., a 99.999% kill in 10 minutes at 25o C) of enteric bacteria, amoebic cysts, and enteric viruses requires > residuals of 0.2, 3.5, and 14.6 ppm, respectively. (2) On a weight basis, iodine can inactivate viruses more completely over a wide range of water quality than other halogens. (3) In the presence of organic and inorganic nitrogenous substances, iodine is the cysticide of choice because it does not produce side reactions that interfere with its disinfecting properties. (4) Iodine would require the smallest mg/L dosage compared to chlorine
,or bromine to "break any water" to provide a free residual. (5) I2 is 2 to 3 times as cysticidal and 6 times as sporicidal as HOI,
while HOI is at least 40 times as virucidal as L. This behavior is explained on the one hand by the higher diffusibility of molecular iodine through the cell walls of cysts and spores and on the other hand by the higher oxidizing power of HOI. Gottardi, W. Iodine and Iodine Compounds in DISINFECTION,
STERILIZATION, AND PRESERVATION, Third Edition, Block, Seymour S.,
Ed., Lea & Febiger, Philadelphia, 1983, and the references cited therein provide more details respecting the background discussed above.
Polyvinylpyrrolidone (PVP, Povidone) is manufactured by BASF Aktiengesellschaft, Untemehemensbereich Feincheme, D-6700 Ludwigshaven,
Germany and sold under the trademark KOLIDON®. Povidone-iodine products and the preparation of such products are described in U.S. Patents 2,707,701, 2,826,532, and 2,900,305 to Hosmer and Siggia, assigned to GAF Corporation and in a number of GAF Corporation publications; see, e.g. Tableting with Povidone USP (1981) and PVP Polyvinylpyrrolidone (1982).
There is extensive patent literature on the manufacture and use of various iodine-polymer complexes, exemplary of which are: U.S. Patent No. 3,294,765, Hort, et al, 1966 - manufacture of povidone-iodine complex; U.S. Patent No. 3,468,831, Barabas, et.al., 1969 - graft co-polymers of N-vinyl pyrrolidone; U.S. Patent No. 3,468,832, Barabas, et.al., 1969 - graft co-polymers of N-vinyl pyrrolidone; U.S.
Patent No. 3,488,312, Barabas, et. al, 1970 - water-insoluble graft polymer-iodine complexes; U.S. Patent No. 3,689,438, Field, et. al., 1972 - cross-linked polymer- iodine manufacture; U.S. Patent No. 3,907,720, Field, et. al., 1975 - cross-linked polymer-iodine manufacture; U.S. Patent No. 4,017,407, Cantor, et. al., 1977 - solid N-vinyl-2-pyrrolidone polymer carriers for iodine; U.S. Patent No. 4,128,633, Lorenz et al, 1978 - preparation of PVP-I complex; U.S. Patent No. 4,139,688, Dixon, 1979 - cross-linked vinylpyrrolidone; U.S. Patent No. 4,180,633, Dixon, 1979 - cross- linked vinylpyrrolidone; U.S. Patent No. 4,190,718, Lorenz, et.al., 1980 -increasing molecular weight of polyvinylpyrrolidone. Under ordinary conditions, PVP is stable as a solid and in solution. The single most attractive property of PVP is its binding capability. This property has permitted utilization in numerous commercial applications. Small quantities of PVP
stabilize aqueous emulsions and suspensions, apparently by its absorption as a thin layer on the surface of individual colloidal particles. The single most widely studied and best characterized PVP complex is that of PVP-iodine. For example, hydrogen triiodide forms a complex with PVP that is so stable that there is no appreciable vapor pressure. It is superior to tincture of iodine as a germicide.
Various poloxamers (i.e., polyether alcohols) also make effective carriers for iodine (i.e., Prepodyne, Septodyne) that exhibit the same germicidal activity as povidone-iodine. The iodophors are available in a variety of forms, such as a 10% applicator solution, 2% cleansing solution (scrub), aerosol spray, aerosol foam, vaginal gel (for trichonomal and candidal infections) ointment powder, mouthwash, perineal wash, and whirlpool concentrate (all 2%). All iodophors may be used in this invention in some of its various uses and applications and, to the extent that the iodophor is effective and does not injure the material undergoing treatment, are considered generally as equivalents or potential equivalents of povidone iodine. In addition to the risk of transmitting infectious disease via blood or blood products, the growth of bacteria in blood and blood products at various stages of production and processing introduces pyrogens into the blood component or product which must be removed before the product can be used in therapy. Introduction of molecular iodine, e.g. povidone- , at an early stage in processing of blood products greatly reduces or eliminates the pyrogen-load of the ultimate product or fraction.
Generally this invention is applicable to the treatment of donated blood and products produced from blood, for inactivating virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms.
Various medical and blood handling procedures are referred to hereinafter. These are all well-known procedures and steps in these procedures are fully described in the literature. The following references are provided for general background and as sources for detailed reference to the literature as to specific procedures: TECHNICAL MANUAL of the American Association of Blood Bankers, 9th Ed. (1985); HLA TECHNIQUES FOR BLOOD BANKERS, American Association of Blood Bankers (1984); Developments in Biological Standardization, Vols. 1 - 57, S. Karger, Basel; CLINICAL IMMUNOCHEMISTRY, The American Association for Clinical Chemistry;
MEDICINE, Vols. 1 - 2, Scientific American, New York; Care of the SURGICAL PATIENT, Vols 1 - 2, Scientific American, New York; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, New York. Summary of the Invention
Safe transfusion blood is manufactured by removing a substantial portion of blood plasma, and optionally coagulating to serum, from whole blood leaving a blood cell concentrate, passing the plasma, or, optionally, serum from such blood, into contact with a solid source of available oxidizing iodine for inactivating pathogenic microbes in the blood and for transferring an effective amount of iodine, from about 0.01% by weight to five percent by weight, into the plasma, or, optionally, serum from such blood, to inactivate pathogenic microbes in both the plasma, or, optionally, serum from such blood, and blood cell concentrate and reconstituting the whole blood by mixing the plasma, or, optionally, serum from such blood, containing available iodine with the blood cell concentrate, the available iodine in the plasma, or, optionally, serum from such blood, being from about 0.01% to about five percent by weight of the thus iodized plasma, or, optionally, iodized serum from such blood, and sufficient to inactivate substantially all pathogenic microbes in the blood cell concentrate. Optionally, other viricidal or biocidal compositions may be added to the plasma, or, optionally, serum from such blood, or to the reconstituted blood to enhance the viral or microbial inactivation.
Description of the Preferred Embodiments The following is given as a basic, exemplary method of carrying out the present invention. It will be understood that the present technique can be adapted to integrating the present invention into overall blood collection and handling technique, that additional steps may be added, and that many variations are possible within the scope of the invention. A unit of transfusion quality blood is separated into plasma, or, optionally, serum from such blood, and blood cell concentrate. Serum may, for example, be formed by coagulating plasma or by coagulating a separate
unit of whole blood from the same donor. Plasma removal may be accomphshed by compressing the blood through a filter to express a substantial amount of the plasma from the container leaving a blood cell concentrate, by centrifuging the blood lightly and decanting the plasma from such blood, or in any other way. Serum may be formed from the plasma or from another unit of the same blood directly by coagulation and centrifugation, or in any other conventional way. Separating a substantial amount of the plasma, or, optionally, serum from blood to form the blood cell concentrate can be carried out using any convenient manipulation(s). It is not necessary that all of the plasma, or, optionally, serum from such blood, be separated; however, best results are achieved when substantially all of the plasma not interstitiaUy trapped between the cells of the cell concentrate is removed.
The plasma, or, optionally, serum from such blood, is then contacted with a sohd source of iodine that holds the iodine loosely enough to permit some of the iodine to enter into the plasma, or, optionally, serum from such blood. One convenient way of carrying out the invention is to express plasma, or, optionally, serum from such blood, through a bed (filter) of polymer- iodine particles. The separation of plasma from blood and iodinazation may be accomphshed by expressing the plasma from whole blood directly through a sohd iodine-containing filter. This provides contact with a sohd source of iodine and helps effect a separation of the plasma, or, optionally, serum from such blood, from the cell concentrate. Any of several forms of sohd povidone-iodine or other polymer-iodine complex is a suitable form of such iodine.
The plasma, or, optionally, serum from such blood, and the blood cell concentrate are reconstituted to form whole blood or blood cells carried in serum. Residual iodine in the plasma, or, optionally, serum from such blood, is present in a sufficient amount, from 0.001 to five percent, typically from 0.1 to 1 percent, by weight, in the plasma, or, optionally, serum from such blood, to kill or inactivate all extracellular virus in the blood cell concentrate and substantially all or all intracellular virus by penetration into the cell. It is
important, for use of minimum amounts of iodine while maximizing the biocidal efficacy of iodine in the reconstituted blood to perform the reconstitution relatively promptly after the iodine is added to the plasma, or, optionally, serum from such blood,. Precise time limits have not been established and the length of delay is not critical. Optimally, the blood cell product will be reconstituted within less than about an hour and, preferrably in less than about a quarter of an hour, after the iodine has been added to the plasma, or, optionally, serum from such blood,. Higher levels of iodine addition permit longer delays in reconstituting the blood or blood cell suspension from the cell concentrate and iodized plasma, or, optionally, serum from such blood. A brief reaction period is required after the reconstitution of the whole blood or serum-blood cell composition. A minimum of about two minutes is necessary, and at least about 15 minutes is desirable. Other biological liquids are treated in an analogous manner. The process results in whole blood or a blood substitute consisting essentially of blood cells suspended in serum from the same blood that is safe, being free of pathogenic microbes, and which is also free of any added chemicals except for iodine, iodide (resulting from reduction of the iodine) All iodine is fairly rapidly reduced by blood constituents to iodide or bound to blood proteins, e.g. albumin, and, thus, non-toxic.
Optionally, after a suitable period of time, from a few minutes to an hour or more, the blood is contacted with a solvent for iodine that has no effect or minimal effect on plasma and the hydrophilic components. Normal alkane, e.g. n-heptane, is a suitable extraction solvent for removing iodine from the blood. Of course, iodine that has been reduced to iodide will remain, largely, in the aqueous phase. Other extraction solvents include analogous homolog of heptane and vegetable oils such as cotton seed oil, corn oil, etc. Any hydrophobic solvent for iodine that is essentially inert biologically and has sufficient density difference from water and respecting which water has a high enough interfacial tension to form a clean separation from the aqueous phase may be used as the solvent
Ji assurance of total conversion of iodine to iodide is desired, the
addition of biologically compatible reducing substances, e.g. reducing sugars, ascorbic acid or ascorbate, sodium sulfite, etc. may be added. The reconstituted blood may be passed into contact with a sohd iodine-adsorbing material, e.g. sohd povidone or starch. Any free iodine will, in such process, be adsorbed and removed from the reconstituted blood. Of course, any combination of techniques may be used.
It is, however, well known and firmly established that iodide are well- tolerated in vivo by nearly all human subjects. The present invention, therefore, obviates objections to the introduction of povidone or other polymeric substances into the blood stream of the patient.
Pathogenic microbes that are inactivated in accordance with this invention include virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms.
Data in the above identified co-pending applications, inter alia, establish the biocidal and virucidal efficacy of iodine compounds.
Practical Application This invention is useful in the preparation of safe transfusion blood for use in animal and human therapy.