Microbial pathogen detecting system and process

A system is provided for receiving, maintaining and processing blood samples for the subsequent separation and testing thereof. A pre-packaged blood sample receiving vessel is provided containing appropriate additives and a quantity of beads or other physical bodies for subsequent lysis, centrifugation, and separation of the sample. An additional pre-packaged arrangement is provided at the lab for the handling and removal of the lysed, centrifuged and separated sample in order to obtain the desired portion of the sample for appropriate laboratory testing procedures.

BACKGROUND AND STATEMENT OF THE INVENTION 
Generally speaking, this invention relates to processes and apparatus for 
receiving blood samples for the subsequent testing of the components 
thereof. More particularly, this invention relates to processes and 
devices for the recovery of bacteria from blood by lysis and 
centrifugation. 
Concentration of organisms by centrifugation has been well known for a 
number of years, such as Mycobacterium from sputum, and the obtaining of 
bacteria from spinal fluids, and so forth. In recent years, developments 
have been made of specific devices for receiving and processing blood 
samples, which devices are configured in such a way and contain certain 
components for the subsequent lysis and separation of the components of 
the blood sample by centrifugation. Representative of these recent 
developments are U.S. Pat. Nos. 4,164,449; 4,131,512 and 3,883,425. 
These developments include using purified saponin, detoxified by either gel 
filtration or passing through a filter, in order to obtain a specific pore 
size through ultrafiltration. The procedures taught in these patents 
include testing for toxicity of saponin prior to its introduction into the 
devices for the subsequent lysis and separation procedures. Representative 
of other arrangements involved in these patents include the use of a 
liquid cushioning agent and an angled surface positioned in the separation 
device in order to provide appropriate separation of the various 
components of the sample, once centrifugation has taken place. 
Difficulties may arise in the use of the arrangements as taught in these 
patents, including a double-ended tube for the introduction and removal of 
components from the tube, in that the layers are separated only by liquid 
cushioning agent type procedures and so forth. Thus, control of the 
removal of the supernatant is difficult due to the necessary manipulation, 
or accidental refluxing by the user, causing further dilution and 
potential loss of organisms prior to the actual separation of the layers 
in the devices. In most instances, the double-ended tube is required. 
With this invention, by contrast, a device is provided for receiving blood 
samples, which device includes certain components in combination with 
physical beads or cylinders configured to prevent disturbing the sediment 
following centrifugation, and during withdrawal of the supernatant. 
Removal of the supernate using a pre-packaged arrangement, in accordance 
herewith, provides simple removal of the supernatant from the centrifuged 
sample with fewer manipulations. Also, prior detoxification and filtering 
of the saponin in the receiving container is not required. 
In addition, the arrangement herein contains a combination of certain 
nutrients which facilitate the growth of organisms in the event of a delay 
in the processing of the sample. The sample may be maintained over a 
period of time between the time the sample is taken and the time when it 
is actually conveyed to a laboratory for testing, thus, removing the 
danger of the organisms to be tested having been destroyed by the contents 
of the container over a period of time prior to any testing thereof. 
Thus, the invention herein includes a pre-packaged evacuated container 
containing non-purified and non-detoxified saponin. This may be obtained 
from conventional sources such as, for example, practical grade saponin 
from Eastman Chemical Company or Fisher Scientific Company. While it is 
appropriate, as will be understood by practitioners-in-the-art, to pretest 
saponin prior to its introduction into the evacuated container because 
batches of saponin will vary, it is not necessary to purify, filter or 
detoxify the saponin prior to its introduction. The inventors have 
discovered, also, that by including certain other additives or nutrients 
in the device of the invention, the growth of organisms is facilitated in 
the event of a delay in processing the sample. Such materials, as 
discussed in more detail below, include, for example, yeast extract or 
other peptones, amino acids and co-enzymes. 
Also included in the container are particles in the form of beads, pellets 
or cylinders which serve to enhance the separation during centrifugation 
of the components of the sample to be tested. Due to the solid or physical 
properties of the beads or other configured particles introduced into the 
evacuated tube for receiving the sample, there is less opportunity for the 
user to disturb the microbial sediment or concentrate during transfer of 
the specimen from the centrifuge to the work area. 
The invention here includes, in addition to the pre-packaged evacuated 
container for receiving the blood sample, a pre-packaged supernate and 
sediment removal unit for use by the laboratory in obtaining the 
appropriate separated components of the sample once lysis and 
centrifugation have taken place. The pre-packaged arrangement includes a 
supernate transfer needle for penetration of the evacuated container 
containing the sample. This needle may be used once centrifugation has 
taken place for withdrawing the supernate from the original sample 
receiving container. The package also includes a vent needle which is 
inserted prior to the removal of the supernate in order to facilitate this 
removal. In addition, the package includes an evacuated sterile container 
for receiving the supernate. Finally, the package includes a needle and 
syringe for removing aliquots of the remaining concentrate in the original 
sample containing container for distribution on appropriate culture 
plates. 
Thus, as purely illustrative of a procedure which may be utilized, in 
accordance with this invention, first blood is collected from a patient by 
drawing directly into an evacuated pre-packaged container. The 
pre-packaged container contains a specific formulation for enhancing the 
maintenance of the sample during a period prior to the sample being 
received and processed in the laboratory. Subsequently, on arrival in the 
laboratory, the sample is centrifuged. A representative centrifuging 
process would be at 3000-3500XG for 30-45 minutes. 
Subsequently, the top of the stopper of the centrifuged container is 
disinfected and the vent needle from the separate packaging, in accordance 
herewith, is introduced into the stopper. Then the supernate transfer 
needle is made to penetrate the stopper. It will be understood that the 
needle is of a sufficient length to be introduced into the container to 
the level of the beads or cylinders or other material utilized for the 
physical separation procedure. A second evacuated container, which is 
sterile, is removed from this package. This evacuated container will have 
sufficient vacuum to draw nine to ten milliliters from the original 
centrifuged container. 
After the supernate is withdrawn, the supernate tube and transfer needle 
are removed. However, the vent needle is left in place. The tube 
containing the supernate, as will be understood, may be removed from the 
transfer unit and stored. The tube containing the beads or cylinders and 
microbial concentrate is then vortexed. Subsequent to this procedure, the 
tube is inverted and the beads are tapped to the stopper end along with 
the solution of concentrate. Then, utilizing the needle and syringe from 
the very same package, the sample is removed and aliquots of the 
concentrate are distributed, as mentioned above, on appropriate culture 
plates. 
As stated above, one of the significiant aspects of this invention is the 
introduction in the sample receiving container of a component which 
maintains and facilitates growth of microorganisms, when present in the 
sample prior to its being received and worked upon by a laboratory. 
Representative formulations include, for example, a yeast extract present 
within the range of between about 0.5-3 grams, and preferably 1 gram, 
sodium polyanethole sulfonate present within the range of between about 
0.8-1.5 grams and preferably 1 gram, a practical grade non-purified and 
non-detoxified saponin present within this range of between about 10 and 
15 grams, and, preferably, 10 grams, and IsoVitaleX.TM., a product of BBL 
Laboratories, Division of Becton Dickinson and Company, Baltimore, Md., 
present within the range of between about 1-3 milliliters, and preferably 
one milliliter, with the above four components dissolved in 100 
milliliters of water. The IsoVitaleX.TM. is a nutrient component 
containing nicotinamide adenine dinucleotide, glutamine, vitamin B12, and 
a sulfur containing amino acid such as cysteine. Added to the above 
formulation is sufficient sodium bicarbonate to adjust the pH to between 
6.9 and 7.4. 
As further illustrative of components which may be added to the evacuated 
container, the formulation above may have added to it Rhozyme.RTM. 
proteases within the range of between about 0.4-1, gram, and preferably, 
0.5 gram. The Rhozyme.RTM. proteases usually selected will be Rhozyme 
41.RTM. concentrate, a product of Rhome and Haas, and which is a 
proteolytic agent. 
Representative formulations for addition to an evacuated container, 
containing the beads or cylinders, for subsequent lysing and 
centrifugation are listed below. 
FORMULA 1 
1 gram: yeast extract 
1 gram: sodium polyanethole sulfonate 
10 grams: saponin-practical grade-nondetoxified 
1 milliliter: IsoVitaleX.TM. 
The above four components are dissolved in 100 milliliters of water and 
approximately 1.6 grams of sodium bicarbonate is added to adjust the pH to 
within the range of between about 6.9 and 7.4. 
FORMULA 2 
1 gram: yeast extract 
1 gram: sodium polyanethole sulfonate 
1 milliliter: IsoVitaleX.TM. 
10 grams: saponin 
0.5 grams: Rhozyme 41.RTM. 
The above five components are dissolved in 100 milliliters of water and 
sufficient sodium bicarbonate is added to adjust the pH to within the 
range of 6.9 to 7.4. 
With respect to the beads or cylinders or other small objects introduced 
into the evacuated container for receiving the sample initially, the beads 
or cylinders may be comprised of plastic or a glass material, and they 
must be selected to be of a size which does not pack and restrict the 
sedimentation of bacteria at the bottom of the tube during centrifugation. 
Examples of satisfactory materials are cylinders having a length within 
the range of between about 3 and 5 mm. and a diameter within the range of 
between about 2 and 3 millimeters. Beads may have a representative 
dimension within the range of between 2 and 5 mm. in diameter. 
Representative materials include polycarbonate such as Lexan, a product of 
General Electric Company, or polytetrafluoroethylene.

DETAILED DESCRIPTION OF THE INVENTION 
Referring to the drawings in which like reference characters refer to like 
parts throughout the several views thereof, FIG. 1 shows the concentration 
system of the invention which is in the form of an evacuated container for 
receiving initially the blood sample. The evacuated container 10 is in the 
form of an elongated tube having an open end 12 and a closed end 14. 
Introduced into the pre-packaged evacuated container, prior to evacuation 
thereof is a representative quantity of particles 18, which may be beads 
or cylinders as discussed above, for the subsequent separation procedures 
once a blood sample is introduced into the container. Also introduced into 
the container is the representative Formula 1 or Formula 2 as noted above. 
The evacuated container size may vary depending upon the amount of 
additive, as long as the total additives content are generally in the 
ratio of 1 part additive to 18-20 parts blood specimen. Thus, an evacuated 
tube will contain about 0.5 ml. additive to draw 9.5-10 ml. of blood 
specimen. The actual quantity of beads, or cylinders introduced into the 
container will be within the range of between about 1 gram and 3 grams, 
depending upon the material used. The column height of the particles or 
beads should be between 1 and 2 inches. 
Once the formula for maintaining the sample has been introduced into the 
container and the particles 18 introduced, the tube is then sealed with a 
conventional evacuated tube stopper 16 and, during the introduction of the 
stopper the evacuation procedure is applied to the tube so that it is 
properly evacuated. This concentration system is pre-packaged for use by 
the medical practitioner or paramedic in taking a blood sample. Once the 
sample is introduced into container 10, it is sent to the laboratory for 
the subsequent procedures, as discussed above, wherein tube 10 is 
centrifuged for a period of time and at a specific rate, as desired. 
Referring now to FIG. 2, a pre-packaged arrangement is provided in the 
laboratory in the form of a centrifugation system for detecting 
bacteremia. This package is the supernate and sediment removal units all 
in a single container for the technician to utilize once the initial 
evacuated container, as shown and described in FIG. 1 has been 
centrifuged. Thus, the evacuated container stopper 16 is disinfected, 
probably with iodine, and the vent needle 50 on holder 54 is removed from 
its shield 51. The vent needle 50 is then inserted in stopper 16. 
Subsequent to this insertion, the supernate transfer needle 32, mounted on 
tube holder 36 has the point 40 thereof inserted into and through stopper 
16 to the level of the beads 18 in tube 10. Then the evacuated tube 41, 
having an open end 44 and a closed end 46, with a stopper 48 closing the 
open end 44, is inserted into holder 36 so that the point 38 of needle 32 
will penetrate through stopper 48. 
Tube 41, as will be understood, has sufficient volume to draw 9-10 
milliliters of the supernate from tube 10. It should be pointed out in 
this respect, that tube 10 may simply be placed in a tube rack for the 
removal of the supernatant material therefrom. It is not necessary for the 
tube to be held at a specific angle for the removal of the supernatant, as 
required in prior art procedures. Moreover, because the tube may be placed 
in a tube rack, it does not have to be maintained at eye level for this 
removal, thus allowing for batch procedures, rather than specific handling 
of each individual tube for the removal of the supernatant component 
contained in the tube. This stabilized procedure also provides for a 
constant volume removal of supernatant from evacuated tube 10, thus 
minimizing the chance of erroneous results. The user avoids other 
manipulation procedures which may cause accidental refluxing partially 
removing supernatants back into the concentrated organism during this 
separation procedure. 
Once the supernatant has been removed, the vent needle is left in place, 
and the tube 41, containing the supernate may be removed from the transfer 
unit and stored. Tube 10, containing the particles 18, as discussed 
previously, and the remaining microbial concentrate are vortexed. 
Thereafter, tube 10 is inverted and the beads are tapped to the stopper 
end along with the solution of the concentrate. Then, the syringe and 
needle apparatus 56 in package 30 may be utilized to remove through 
stopper 16 the concentrate. Thus, shield 58 is removed from needle 60 of 
the syringe and needle unit 56, and the handle 66 is utilized to draw 
plunger 62 to remove the concentrate from tube 10 through stopper 16. 
Aliquots of this concentrate contained in syringe 56 may then be 
distributed on appropriate culture plates, as desired. It will be noted in 
FIG. 2, that the package 30 is sealed in the usual sterile package 
arrangement which may be pealed open at 31, as desired. It will be 
understood, that package 30 is desirably comprised of a flexible clear 
plastic material so that contents thereof are clearly visible for the 
technician. 
As discussed previously in this specification, the particular system, in 
accordance herewith, utilizes an additive introduced into evacuated 
container 10 for receiving the initial sample. This additive has the 
effect of maintaining the sample in appropriate condition for a relatively 
long period from the time the sample is received in the container until 
such time as the laboratory has time to work upon the sample. As will be 
appreciated, this is a necessary provision because many doctors take 
samples from their patients in the office and a certain amount of time is 
taken from receipt of the sample until such time as the laboratory 
technician has an opportunity to lyse and centrifuge the sample for 
subsequent procedures. Thus, it is helpful if the sample may be maintained 
for as long as 18 to 24 hours without having a deleterious effect upon the 
organisms contained in the sample. A representative comparison was made 
utilizing the invention herein containing Formula 2 and three different 
organisms were tested. As a comparison, an ISOLATOR.TM. brand unit was 
tested also. The ISOLATOR.TM. brand unit is configured in accordance with 
the teachings of the patents noted above in this specification. Table 1 
below shows the comparisons made during this test procedure. 
TABLE I 
__________________________________________________________________________ 
Unit held 18-24 hours before centrifugation at 35.degree. 
C. 
"0" time 24 hours 
Organisms 
Unit tested 
CFU/tube 
-x 
CFU Supernatant 
Sediment 
__________________________________________________________________________ 
L. monocytogenes 
A 595 .+-.87.8 
tntc CF 
B 595 0 0 
S. pneumoniae 
A 13 .+-.4 
tntc CF 
B 13 0 0 
S. pyogenes 
A 126 .+-.8.4 
tntc CF 
B 126 0 0 
__________________________________________________________________________ 
-x = average of 5 plates: 0.3 ml/plate 
CF = confluent growth (&gt;1000 CFU) 
SD = Standard Deviation 
tntc = too numerous to count on plate (&gt;500 CFU) 
CFU = Colony Forming Units 
A -- Unit according to invention 
B -- Dupont Brand ISOLATOR .TM. Unit 
As can be seen in Table 1, organisms were destroyed using the ISOLATOR.TM. 
brand unit after a 24-hour period while the organisms were maintained 
stabilized for subsequent testing with the unit of the invention, in 
accordance herewith. Indeed, each of the three organisms compared, were 
too numerous to count on the plate and having greater than 500 colony 
forming units at least in the supernatant, and having confluent growth of 
greater than 1000 colony forming units in the sediment. 
Thus, as will be appreciated, there is provided in accordance with this 
invention a system and a procedure for receiving a blood sample containing 
bacteria which system provides for the subsequent lysis and centrifugation 
of the blood sample to obtain the desired bacteria for subsequent testing. 
The system includes a formula for maintaining the sample over a long 
period of time between the time the sample is taken, and the time the 
laboratory technician has time to obtain the sample and to make 
appropriate processing procedures to the sample in order to obtain the 
desired number of culture plates of the bacteria contained. Moreover, the 
process and package of the invention includes a complete sterile package 
of components all necessary for carrying out the procedure for separating 
and removing the supernatant and concentrated components of the blood 
sample, once the sample has been received by the laboratory technician for 
centrifugation. As will be appreciated, the arrangement is such that the 
samples are in appropriate condition for proper testing with sufficient 
bacteria to carry out the proper testing procedures. 
While the systems and methods herein described constitute preferred 
embodiments of this invention, it is to be understood that this invention 
is not limited to these precise methods and systems of apparatus, and that 
changes may be made therein without departing from the scope of the 
invention which is defined in the appended claims.