BIOLOGICAL FLUIDS AND CONTAINER FOR SAME

The present invention iβ directed to a device and method for combating and destroying biological contamination in specimens of biological fluids such as blood intended for medical evaluation without interfering with the integrity of the proposed evaluation. More specifically, the present invention iβ particularly concerned with disinfecting viral co tamination in biological specimens to avoid infecting those coming in contact either with the specimen itself or the receptacles and equipment used to contain and evaluate the specimen. Of particular concern in the present invention is the avoidance of contamination by HTLV-III Virus responsible for Acquired Immune Deficiency Syndrome and Hepatitis Virus which may be present in blood specimens drawn for medical evaluation.

BACKGROUND OF THE INVENTION

The incidence of hospital acquired infections has been increasing in recent years at an alarming rate which has caused great concern among the staffs of hospitals and especially those working in the laboratories. While many disinfection and sterilization techniques have been employed to alleviate this problem in different functional sections of the hospital, th_se techniques have not consistently provided a safe environment for the staff. Frequently, the disinfection and sterilization techniques which have been used have been employed after overt contamination has taken place such a s spilling, broken samples, etc. While these techniques have helped to reduce the incidence of laboratory acquired infections, they have not curtailed them. With the increasing incidence of contagious pathogens that can be transmitted by patient's specimens, especially blood and particularly such dangerous contaminants as the AIDS and hepatitis viruses., a new and safer technique for handling labora tory specimens is needed.

Various disinfectants and sterilizing agents have been employed with varying degrees of success, both in hospitals and other environme ts. Monoa ldehydes such as

formaldehyde have been used successfully as a disinfectant, however, dialdehydes, particularly glutara ldehyde, have been more preferred. Examples of glutara ldehyde-ba sed disinfectants are a dilute sodium phena te-glutara ldehyde solution buffered to pH 7.4, an activated solution which contains 2.0% glu tara ldehyde buffered to pH 7.5-8.0 and a disinfectant and sterilizing solution containing 2% glutara ldehyde at pH 7.0-7.5.

The extensive use of glutara ldehyde based compositions as an antiseptic and disinfectant has led to extensive studies of the compound and its activity. Glutaraldehyde has been classified as a chemos terilizer and has been defined by Borick, J. of Pharm. Sciences, vol. 53, no. 10, October, 1964, as a chemical agent capable of destroying all forms of microbiol life including bacterial and fungus spores, tubercle bacilli and viruses. The compound has in fact been shown to be effective against a wide range of viruses even in the presence of high levels of organic matter which tend to destroy the potency of other disinfectants. The degree of biocidal activity observed in glutara ldehyde solutions is very much dependent on the pH of the solution as enhanced biocidal activity is found in alkaline solutions.

Boucher et al., Proc. Wes t Pharmaca 1 Soc. 16, pp.282-288, 1973, postulated that the biocidal activity of glutar ldehyde is controlled by the distance between the aldehyde groups and their tendency- to polymerize thereby allowing free aldehyde groups to interact with the amino groups, of the bacterial cell. This agrees with the findings of Rubbo et al., J. Appl. Bacteriol 30, pp.78-87, 1967, that antibacterial activity is due to the two aldehyde groups present on the molecule. After considering these results, Navarro and Monsan, Ann. Microbiol 12 _^ 7B, pp.295-307, 1976, concluded that only structures containing two aldehyde groups allow formation of an aldol type polymer at an alkaline pH, and also produces a similar sterilizing effect at acid pHs on increasing concentrations. In other words, while the extent of polymerization is considerable at

alkaline pHs, it is negligible in acid solutions unless the concen ration is increased. On the other hand, acid solutions at pH3-4 of glutaraldehyde are considerably more stable than alkaline solutions.

The antimicrobial activity in any compound can not be viewed in isolation but must be described with reference to a number of factors including pH, temperature, organic matter present, and concentration. For glutara ldehyde, it h s been common to use a 2% solution at room temperature and an alkaline pH of about 7.9. Unfortunately, alkaline solutions of glutaraldehyde are much less stable than acid solutions owing to the polymerization reactions already described, with a corresponding loss of antimicrobio1 activity. A reduction in sporicidal activity of activated glutara ldehyde on storage h s been observed in reports of

Kelsey et al., J. C1in._ Pa thoi 2J, pp.632-638, 1974, Thomas and Russell, J. Appl. Microbiol 2j3, pp.331-225, 1974b,

Gorman and Scott, Int. J. Pharma 4, pp.57-65, 1979a. This reduction in sporicidal activity is directly related to a drop in concentration of the free aldehyde which appears to be essential for biological activity. Borick, Adv. Appl.

Microbiol 1_0, pp.291-312, 1968, h s estimated that glu tara ldehyde concentration actually falls from 2.1% at pH 8.5 to 1.3% at pH 7.4 over a period of twenty-eight days at ambient temper tures. Accordingly, it has generally been the practice to employ glu tara ldehyde as a 2% solution to which an activator is added to bring the pH to approximately 8 at the time of use. Such a solution used at room temperature will, for example, disinfect within 10 minutes and sterilize within 10 hours. However, it has been recommended that this solution be discarded after 14 days because of the significant decrease in activity and free aldehyde concentra ion. This instability has led to the development of more stable preparations formulated at lower pHs and some with other potentiators included to increase the otherwise low level of activity observed at lower pH.

The inevitable conditions of clinical use for disinfection and sterilization frequently mean that organic

matter is present such as blood and pus. This organic matter can act either by protecting the microbial species from antimicrobial attack or by competing with the microbial cell for active sites on the disinfectant molecules, thus reducing the effective concentration of disinfectant substance. Accordingly, many otherwise effective disinfectan s and sterilizing agents may become ineffective where organic material, such as blood, is contacted.

Glutara ldehyde , however, has a high resistance to neutralization by organic matter. Borick et al., J. Pharm.

Sci. 53, pp .1273-1275, 1964, for example has reported that the presence of 20% blood serum did not appear to adversely effect the activity of glutaraldehyde while Snyder and

Che tle, Am. J. Hosp. Pharm. 22, pp.321-327, 1965, have reported that 1% whole blood did not effect glu tara ldehyde activity.

One of the most important considerations in selecting a suitable disinfectant, in addition to its potency and sustained effectiveness as a disin ectant, is the toxicity of the composition to individuals coming in contact with it. Various studies have shown that glu tara ldehyde, in moderate effective concentrations, is generally only slightly irritating to the skin, mucous membranes and eyes. Sato and Dobson, Arch. Derma tol 100, pp.564-569, 1969, have found that 5% glu tara ldehyde was only irritating if the epidermal barrier was not intact.

Aqueous solutions of glu tara ldehyde have been used to treat hyperhydrosis and it has been used topically in the treatment of onychomycosis . Prevention of dental calculous formation and reduction of dental cavity formation in the mouth has been achieved by using oral compositions incorporating glu tara ldehyde . In the cosmetic field, glu tara ldehyde has been proposed for disinfection of production equipment and as a preserv tive. Glutara ldehyde has been used as a disinfectant for control of mastitis.

Accordingly, glu tara ldehyde is now a generally accepted disinfectant and is found in a number of commercial preparations for disinfection and s erilization. Babb et

al., J. Hosp . Infec. 1, pp.63-75, 1980, for example, have compared nine glu ta ra ldehyde products.

Glutara ldehyde has also been used extensively in various non microbiological areas including the leather tanning industry and tissue fixation for elee tromicroscopy. In microbiological areas, glu tara ldehyde has been employed principally as a liquid chemical sterilizing agent for medical and surgical material that cannot be sterilized by hea or irradiation. Compared with other disinfectants, glutara ldehyde has been found to be superior for disinfection of face masks, breathing tubes and other respiratory therapy equipment. Important advantages of glu tara ldehyde as a chemos terilizer are: its activity in the presence of organic material, non-corrosive action towards metals, rubber, lenses and most materials, and lack of deleterious effect on cement and lenses of endoscopes. Further, glutara ldehyde has been recommended for decontamination of dental, surgical instruments and working surface where the hepatitis B surface antigen may be present as well as for the treatment of warts.

From the above mentioned studies testing any biological specimen containing glu tara ldehyde will not damage the instrument used in testing. Osterberg, Arch.

Pharm. Chemi. Sci. Ed. 6, pp.241-248, 1978, found that damage to leukocytes was apparent only above a 100 microg/ml. glu tara ldehyde level. In addition, no erythrocyte damage occurred at the glu t ra ldehyde concen rations used.

The use of aldehydes in electron microscopy was extensively studies and it was found that many cytochemical reactions can be performed on tissue specimens after aldehyde fixation. Glutara ldehyde is effective in preserving both prokaryotes and eukaryotes, including fragile specimens such as marine invertebrates, embryos, diseased cells and fungi. Glutara ldeh de stabilizes blood plasma with little shrinkage of blood clots (Chambers et al. 1968, arch. Pathol. 85,18.). Tissue specimens can be left in this fixative for many hours without apparent

deterioration. Presently, glu tara ldehyde is the most efficient and reliable fixative for preservation of biological specimens for routine electron microscopy and the previously mentioned and available data indicate that proteins are not denatura ed to any marked extent by fixation with glutara ldehyde (M.A. Hayat, Fixative for elee tromicroscop , Academic Press, 1981). Similarly, glutara ldehyde fixed-erythrocy tes remain sensitive to the hemagglu ina tion and hemagglutina tion Inhibition tests for arbovirus antigens and antibodies (Wolff et al. [1977] J.

Clin Microbiol. 6.55). Differential staining of viable and nonviable cells with alcian blue is maintained after fixation with glu tara ldehyde (Yip and Auerperg, 1972, In

Vitro 7, 323). From the above mentioned studies, glutara ldehyde will preserve the biological specimens without otherwise affecting the Integrity of the specimen for future evaluation.

As set forth above, the handling of biological specimens such as blood after sampling, during storage and medical evaluation poses a particular hazard for those coming in contact with the specimens, especially where there is a possibility of AIDS (HTLV-III) or Hepatitis Virus being present. Despite the known effectiveness of disinfectants such as glutara ldehyde in destroying these viruses, their use has essentially been limited to the containers and equipment coming in contact with the fluid, and only after such contact has occurred and the fluid disposed of. What remains especially hazardous is the contaminated body fluids themselves, such as AIDS (HTLV-III) or Hepatitis infected blood, which are carriers of the infection from the time they are drawn from the donor. Accordingly, what is needed is a technique for destroying such viral contamination instantaneously when the sample is taken, but without effecting the specimens for further testing.

DISCUSSION OF THE PRIOR ART

U.S. Patent Number 3,016,328 describes disinfecting with a sporicidal composition containing a C2

^t0 ^ saturated dialdehyde, such as glutara ldehyde, and an alkalinating agent In either alcoholic or aqueous solution a t a pH above 7.4.

U.S. Patent Number 3,282,775 describes disinfecting with a sporicidal composition containing a C2 to C, saturated dialdehyde preferably glutara ldehyde and a cationic surface active agent.

U.S. Patent Number 3,708,263 describes sterilizing at temperatures below 75 C by contacting the equipment to be treated with an aqueous solution at pH 2 to 8.5 containing glu taraldehyde and DMSO simultaneously with ultrasonic wave energy.

U.S. Patent Numbers 3,912,450; 3,968,248; and 3,968,250 describe disinfection or sterilization compositions that contain nonionic and anionic surfactants with aqueous or alcoholic glutara ldehyde solutions.

U.S. Patent Number 4,093,744 describes sporicidal compositions containing glu tara ldehyde at pH 6.5 to 7.4 which may contain a detergent and also a monoa ldehyde.

U.S. Patent Number 3,983,252 describes disinfectant compositions that contain a dialdehyde and an alkaline metal salt of a hydrocarbon carboxilic acid in aqueous solution and optionally an alcohol of up to seven carbon a oms or a diol with up to 4 carbon atoms such as ethylene glycol, propylene glycol, butylene glycol and/or a triol glycerol. The compositions are described as having improved stability in the pH range of 6 to 7.4.

U.S. Patent Number 4,103,001 describes a sterilizing composition containing glu tara ldehyde , a phenol and a metal phenate as active ingredients. The composition may also contain a humectant such as glycerol, propylene glycol or diethylene glycol.

U.S. Patent Number 4,436,754 describes a disinfectant and sterilizing composition having low odor and irritation potential which is an aqueous solution containing a 2 to 6 carbon a torn dialdehyde and may also contain formaldehyde and a diol or mono-substituted diol. Such compositions can be used at a pH of 2 to 9.

U.S. Patent Number 3,886,269 describes a formaldehyde based disinfectant formed by passing formaldehyde gas through a solvent such as dimethyl sulfoxide or dimethyl formamide to form a gel-like polymer.

The disinfectant described exhibits disinfection properties against bacterial vegetative cells, bacterial spores, and soil organisms .

U.S. Patent Number 4,048,336 describes the use of a combination of glu tara ldehyde and a monoaldehyde such as formaldehyde to kill spores on instruments.

M.A. Hayat in Fix tion _ for ^le ^c tromicroscopy, Academic Press, 1981, pages 64 to 147 describes fixative agents for preserving and fixing blood and/or tissue specimens .

Seymour S. Block in Pis infec tion, S teriliza tion and Preserva tion, Lea and Febiger, 1983, Chapters 2, 3, 9 and 22 describes sterilization techniques using glutara ldehyde and phenolic compounds.

DESCRIPTION OF THE INVENTION

In accordance with the present invention, a disinfectant for viral and other contamination in biological fluids such as blood is provided in a container for the biological fluid in an amount which is effective to destroy the contamination without otherwise compromising the integrity of the fluid specimen with regard to subsequent biomedical evaluation. The present invention is particularly adapted for use with evacuated containers into which freshly drawn specimens of blood are introduced and held for subsequent study. Such containers typically consist of a cylindrical tube having one open end into which an elastomeric stopper is fitted which is capable of accepting a hollow syringe needle to permit introduction of the biological fluid into the tube. Vessels of this sort are commercially available under the name Vacutainer Systems from Bee ton-Dick inson for example and are evacuated to provide a partial vacuum and provided with a hollow syringe needle which is disposed so that blood is drawn from the donor into the tube by the force of the vacuum in the tube.

According to the invention, the receptacle for receiving and holding the specimen of a biological fluid such as blood is provided with a disinfectant prior to introduction of the biological fluid in an amount sufficient to destroy viral contamination in the fluid and the receptacle without compromising the integrity of the specimen for medical evaluation. The disinfectant is preferably a mono or dialdehyde such as either glu tara ldeh de or formaldehyde or a combination thereof, with the glutara ldehyde being the most preferred. The effective concentration of glut ra ldehyde according to the invention is about 0.1 to 2.5 weight percent, preferably 0.13 to 2.0 weight percent based upon the total quantity of biological fluid to be placed in the receptacle. Thus, the actual amount of the glu tara ldehyde present in the receptacle before introduction of the fluid will depend on the size of the receptacle and the extend to which it is to be filled with fluid since the fluid is, in effect, the principal dilutent. Lesser concentrations of glu ara ldehyde will have a diminished effectiveness in destroying viral contamination while higher than 2.5% concentrations can effect biomedical evaluation of the fluid. Additional aldehydes such as formaldehyde can also be used with the dialdehyde in amounts of about 0.1 to 3 percent by weight based on the total biological fluid. Where glu tara ldehyde is the disinfectant employed in accordance with the invention, it is desirable to maintain a slightly alkaline pH of preferably about 7.2 to 8.5 preferably 7.4 in order to achieve maximum effect against viral co aminants.

As demonstrated in the prior art, however, glutara ldehyde undergoes increasing polymerization at alkaline pHs and the glu tara ldehyde should be maintained at acid pH until just before use. While the receptacle can be provided with an alkalinating agent such as sodium bicarbonate, sodium phenate, lower alkanols, phenol or quaternary ammonium compounds which is isolated from the glu tara ldehyde until just before introducing the biological fluid, it is preferred according to the invention to

increase the pH of the glu taraldehyde by introduction of the blood specimen itself which has a pH of about 7.4 normally.

Where buffering to a higher pH is required, suitable amounts of alkalinating agent can be used.

It is also desirable to incorporate into the receptacle of the present invention effective amounts of substances to increase the permeability of the cell membrane to, allow the disinfectant to reach intrace1lular pathogins more quickly. Such substances are dimethyl sulfoxide, and glycerol, either alone or in combination. Additionally, other substances whose use in connection with sampling and testing of biological fluids, such as blood, is known can be used such as anticoagulents , preservatives and biocidal agents. By employing the various configurations which are embodiments of the present invention, activation of the disinfectant can take place prior to, during or after introduction of the specimen and the disinfectant can be released either before, during or after the specimen is introduced. The present invention will however be more fully appreciated by having reference to the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates one embodiment of the invention in which a closed tube Is used having a stopper in one end and containing a disinfectant and activator.

Figure 2 illustrates an additional embodiment of the present invention whereby both ends of the tube are stoppered and one stopper is provided with the disinfectant or activator.

Figure 3 illustrates an embodiment of the present invention similar to that of Figure 2 in which one stopper contains anticoagulent.

Figure 4 illustrates an embodiment of the present invention similar to that of Figure 1 except for the presence of an inert barrier material.

Figure 5 illustrates an embodiment of the present invention also similar to that of Figures 1 and 4 in which the stopper contains activator and disinfectant separated from each other.

u

Figure 6 illustrates an embodiment similar to that of Figure 5 having an anticoagulent rather than an inert barrier material.

Figure 7 illustrates an embodiment of the present invention having a tube similar to that of Figure 2 in which one stopper contains activator and disinfectant separated from each other and containing anticoagulant.

Figure 8 illustrates an embodiment of the present invention having a tube similar to that of Figure 1 but containing a disinfectant on the walls of the tube without activa tor.

Figure 9 illustrates an embodiment of the present invention similar to that of Figure 8 except that activator is contained in the stopper.

Figure 10 illustrates an embodiment of the present invention similar to that of Figure 2 but with disinfectant on the inner walls of the tube.

Figure 11 illustrates an embodiment of the present invention in which a stopper is used which contains disinfectant and having a permeable membrane.

Figure 12 illustrates an embodiment of the invention similar to that of Figure 2 except that no activator is used in connection with the disinfectant and a stopper is used having a permeable membrane.

DETAILED DESCRIPTION OF THE DRAWINGS

Directing attention to the drawings, Figure 1 illustrates an embodiment of the present invention in which a cylindrical tube 20 closed at one end is provided with an elastomeric stopper 21 at the other end. As previously noted, closed stopper tubes of similar construction are commonly employed for collecting samples of blood. It is frequently the case that these tubes are provided with a partial vacuum and a double ended hollow syringe needle placed in the stopper end so that the blood sample can be drawn directly from the donor into the tube using the vacuum in the tube. Although the details of construction of these syringe devices is not herein illustrated since they are

well known in the art, it will be understood that they can be used in connection with the present invention. In accordance with the embodiment of the invention shown in

Figure 1, a disinfectant material 23 is predisposed in the bottom of the tube 20 and a suitable alkaline activator 22 such as sodium bicarbonate is provided in a cavity 24 of the stopper 21. The two materials are thus kept separate from one another until the blood sample is introduced through the stopper into the tube whereby the mixing of the glutaraldehyde and activator takes place. It will be understood that the amount of glutara ldehyde present in the bottom of the tube 20 will depend upon the size of the tube and the quantity of blood to be drawn into the tube and should be sufficient to insure a concentration of between

0.1 and 2.5% glutara ldehyde once the blood sample is in the tube. The amount of activator present in the stopper cavity

24 will be sufficient to insure that the specimen and glu tara ldehyde have an alkaline pH between 7.2 and 8, preferably about 7.4.

In the embodiment of the invention shown in Figure 2, the cylindrical tube 25 is provided with a stopper at either end. The lower end of the tube 25 is closed by elastomeric stopper 27 having a recess which contains an activator such as sodium bicarbonate 30 which is separated by thin membrane from glutara ldehyde disinfectant 31 which is disposed freely in the tube. The other end of the tube is closed by stopper 26. A sharp pin 29 having a head 28 is provided for piercing the membrane separating the activator and disinfectant before or once the blood sample has been introduced into the other end of the tube 25 through stopper 26.

Figure 3 of the drawings illustrate an embodiment of the invention similar to that of Figure 2 except that the upper end of the tube 25 is provided with a stopper 32 having a recessed area 33 provided with an anticoagulant separated from the disinfectant to maintain the fluidity of the blood sample. Introduction of the blood sample through the stopper 32 releases the anticoagulant by rupturing a

barrier to allow it to mix with the blood sample, disinfectant and activator which are released by the means of a pin.

In Figure 4 of the drawings, an embodiment of the invention otherwise similar to that of Figure 1 is illustrated in which an activator 39 is provided in the cavity 38 of stopper 37 in the top of the tube. The glu taraldehyde disinfectant is however mixed with an inert barrier material and placed at the bottom of the tube 36. In this manner, activation of the glutara ldehyde to the appropriate pH will not occur until the blood sample is centrifuged to produce a separation of the serum.

In Figure 5 of the drawings, the stopper 40 is provided with a recess 43 containing the activator 41 and disinfectant material 42 which are separated from one another by a thin membrane and from the inside of the tube. Inert barrier material is provided at the bottom of the tube 36.

The embodiment of the invention shown in Figure 6 is similar to tha of Figure 5 except that the inert barrier material is replaced with an anticoagulent 45.

Figure 7 of the drawings illustrates an additional embodiment of the invention whereby stoppers are provided at both ends of the tube 25. The stopper 27 closing the lower end of the tube is provided with an activator at 30 and disinfectant 31 separated from one another by a thin membrane and from the inside of the tube. Anticoagulent is placed in the tube directly over the stopper and disinfectant material. A pin 29 with head 28 is available to puncture the separating membranes to permit the materials to mix with the blood introduced through stopper 26 at the other end of the tube.

Figure 8 of the drawings illustrates a preferred embodiment of the invention in which disinfectant material 50 is coated on the inside of the tube 20 to provide a layer. The upper end of the stop of the tube 20 is closed by stopper 26 but no additional activator is provided since the amount of disinfectant 50 is adjusted so that its pH

will become slightly alkaline with the introduction of blood into the tube which also provides the necessary dilution to result in a concentration of 0.1 to 2% glutara ldehyde.

In Figure 9 of the drawings, an embodiment of the Invention is shown similar to that of Figure 8 in that the disinfectant material is a coating 50 on the inside of the tube 20. An activator such as sodium bicarbonate is provided and separated from the inside of the tube, however, in cavity 38 of stopper 37 at 39.

Figure 10 of the drawings illustrates the embodiment of the invention whereby the cylindrical tube 25 Is closed at both ends by respective stoppers 26 and 27. The stopper 27 is however provided with activator 30 which is separated from the inside of the tube and released into the tube to interact with the disinfectant 50 by inserting the pin 29 into the stopper 27 to rupture a membrane that separates the activator from the interior of the tube.

In Figure 11 of the Invention, either an anticoagulent or activator 51 is provided in the bottom of the tube 44. A porous material container 54 is provided on stopper 52 to hold the disinfectant 53 and permit it to diffuse through a permeable membrane Into the tube 44 once the fluid specimen has been introduced into the tube and the tube inverted. In Figure 12 of the drawings, the disinfectant material 57 is provided in an appropriate cavity in stopper 55 closing one end of the tube while stopper 56 closes the other end of the tube. A membrane prevents the disinfectant from entering the tube itself until blood is introduced, at which time the disinfectant diffuses through the membrane into the specimen.

It will be understood that while various preferred embodiments of the present invention have been described herein in order to illustrate and disclose Applicant's invention, additional variations and applications of the present invention are considered to fall within the scope thereof .