A method and system is disclosed for introducing anti-bacterial
agents, for example, quinolone carboxylic acid derivatives into
blood bag systems for preventing massive bacteria growth in stored
blood and blood components. The quinolone carboxylic acid derivative
can be added to a collection container before, during or after the
collection of blood and blood components. The addition of a quinolone
carboxylic acid derivative to blood bag systems allows for increase
in storage time for blood and blood components without the threat
of massive bacterial contamination.
What is claimed is:
1. A method for reducing the likelihood of bacterial contamination
in stored platelets comprising the steps of:
(a) withdrawing blood from a donor;
(b) collecting platelets from the blood into a collection container;
(c) storing the platelets;
(d) wherein the improvement comprises admixing the platelets with
a sufficient amount of an antibacterial agent to inhibit the growth
of gram negative organisms and gram positive organisms during the
storage of step (c).
2. The method of claim 1 wherein said antibacterial agent is a
quinolone carboxylic acid derivative.
3. The method of claim 2 wherein said quinolone carboxylic acid
derivative is selected from the group consisting of ciprofloxacin,
ofloxacin, norfloxacin and enoxacin.
4. The method of claim 3 wherein said quinolone carboxylic acid
derivative is ciprofloxacin.
5. The method of claim 4 wherein the ciprofloxacin is present in
a final concentration of about 1 .mu.g/ml to about 20 .mu.g/ml.
6. The method of claim 5 wherein the final concentrate of ciprofloxacin
is about 5 .mu.g/ml.
7. The method of claim 1 wherein said antibacterial agent is placed
in a separate but potentially communicating satellite compartment
attached to said collection container.
8. The method of claim 1 wherein said anti-bacterial agent is in
a liquid formulation.
9. The method of claim 1 wherein said anti-bacterial agent is in
a dry formulation.
10. The method of claim 8 wherein said antibacterial agent is a
quinolone selected from the group consisting of ciprofloxacin, ofloxacin,
norfloxacin and enoxacin.
11. The method of claim 9 wherein said antibacterial agent is a
quinolone selected from the group consisting of ciprofloxacin, ofloxacin,
norfloxacin and enoxacin.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the storage of blood and blood components.
It has among its objects a novel system and method of storing blood
and blood components in the presence of antibacterial agents to
prevent massive bacterial contamination. A specific group of such
antibacterial agents include quinolone carboxylic acid derivatives.
2. Description of the Prior Art
Proliferation of inadvertently introduced bacteria into blood and
blood component collection and storage systems is a rare event.
Additional contamination is thought to occur most likely at the
time of phlebotomy. But the consequences of bacterial contamination
of blood and blood components can result in a potentially life threatening
Refrigeration of collected blood and blood components and short
term storage have been found to minimize the incidence of massive
bacterial contamination. Shortening the storage period often results
in the disposal of a large number of units of blood and blood components
that are not contaminated. This results in a loss of an important
and often critical supply of blood and blood products. In addition,
some blood components, such as platelets stored as platelet-rich
plasma and platelet concentrates (PC) possess a better in vivo half-life
when prepared and stored at ambient temperature. With the introduction
of blood bag systems which include film that allow for greater gas
transmissibility, platelets have been stored for periods of up to
7 days. When the storage period was increased to 7 days the incidence
of contamination of stored platelets increased prompting the FDA
to decrease platelet storage from up to 7 days to up to 5 days.
See FIG. 1 showing an increase in the incidence of contaminatin
since the end of 1984.
SUMMARY OF THE INVENTION
The invention described herein is a method and system for preventing
massive bacterial contamination in collected and stored blood and
blood component products by introducing an antibacterial agent into
the collection and storage containers. In the present method and
systems, anti-bacterial agents, such as quinolone carboxylic acid
derivatives can be introduced into the blood bag system before,
during or shortly after collection.
The primary advantage of the present invention is preventing massive
bacterial contamination when blood and blood components are stored
at room temperature or inadvertently not timely refrigerated.
Another advantage of the invention is to increase the storage time
of blood and blood components.
Yet another advantage of the invention is to increase the storage
time of platelets at ambient temperature to greater than 7 days.
This would provide a safer platelet transfusion product by preventing
massive bacterial growth in platelets that are accidently contaminated.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the number of septic reactions/year from platelet
FIG. 2 is a graph of the distribution of minimum inhibitory concentrations
FIG. 3 is a series of graphs of ciprofloxacin platelet data compared
with historical data.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In a preferred embodiment the quinolone carboxylic derivative is
a fluoro-quinolone which is a subset of a general family of quinolones.
Examples of quinolones and their manufacturer include ciprofloxacin
by Miles, ofloxacin by Hoeschst, norfloxacin by Merck, Sharpe and
Dohme, and enoxacin by Warner-Lambert.
Quinolones, such as ciprofloxacin, act by inhibiting the bacterial
enzyme DNA gyrase. Bacterial DNA is a double stranded circle in
which the two strands are wrapped around each other in a helical
fashion. DNA replication is initiated at one point on the circle.
The strands are first pulled apart at the initiation point (to allow
the sequence to be read). This causes the number of twists per unit
length to increase on both sides of the initiation point. DNA gyrase
decreases the twist density and thus allows the continual unravelling
of the entire double stranded circle. Without DNA gyrase, DNA replication
is not possible, and therefore bacterial replication cannot occur.
Ciprofloxacin also has other less understood actions that will
kill bacteria when added at levels higher than the amount needed
to inhibit bacterial replication.
Ciprofloxacin is a broad spectrum antibacterial agent, affecting
both gram negative and gram positive bacteria. It is effective at
low concentrations e.g. 5 .mu.g/ml. It is autoclavable and stable
in plasma at room temperature for 7 days. It is not toxic to humans
by the intravenous (IV) route at levels (100-200 mg) that are 50
to 100 times the dose that would be received in a transfusion of
eight 50 ml PC each containing 5 .mu.g/ml ciprofloxacin.
Experiments to Show the Effectiveness of Ciprofloxacin
There are a number of published reports showing the effectiveness
of ciprofloxacin e.g. Wolfson JS and Hooper DC. The Fluoro-quinolones:
Structures, Mechanisms of action and Resistance, and Spectra of
Activity in vitro. Antimicrob. Agents Chemother. 28: 581-6 (1985).
A number of experiments to confirm ciprofloxacin's effectiveness
has been performed. Nineteen different organisms were isolated from
skin at the phlebotomy site from 11 individuals representing multiple
nationalities and races (Chinese, black, white, Korean). Sterile
Q-tips were dipped in sterile saline and rubbed vigorously at the
phlebotomy site. The entire surface of an agar plate containing
an all purpose growth medium (5% sheep blood trypticase soy agar)
was rubbed with the Q-tips. The plates were incubated for 2 days
at 37.degree. C. One to 3 different colony types were found per
plate. Representatives of each colony type were subcultured at 22.degree.
C. to select for organisms that would grow at the temperature in
which platelets would be stored. Further culturing was done to yield
pure strains. The strains were characterized by morphology, gram
stains, growth on mannitol salt agar, ability to grow anaerobically
on glucose, and the coagulase test. All strains were gram positive
cocci and appeared singly or in clusters. Four were identified as
Micrococcus by their inability to grow anaerobically on glucose.
Fourteen strains were identified as staphylococci by their ability
to show good growth anaerobically on glucose. One strain remained
unidentified at the group level by showing only weak growth on glucose
anaerobically. Three strains were identified tentatively as Staphylococcus
aureus by a coagulase positive result, although the results were
tentative and conflicted with the results of growth on a mannitol
For each of the 19 human strains the minimum concentration of ciprofloxacin
to completely inhibit growth (MIC in .mu.g/ml) was determined. Also
included were S. aureus ATCC 14514 and S. epidermidis QA. The MIC
tests were performed on agar plates containing Mueller-Hinton medium.
A series of plates were made containing ciprofloxacin in the medium.
The range of concentration varied 2 fold from 10 .mu.g/ml to 0.038
.mu.g/ml. On each plate in a known spot (usually 9 to 10 strains
per plate), 50000 colony forming units (CFU) in 5 .mu.l were added.
The experiment was run in duplicate. The plates were incubated overnight
at 37.degree. C. The MIC is defined as the concentration of ciprofloxacin
in which no growth is observed. The results for the 19 human isolates
and 2 reference strains of gram positive cocci are shown in Table
1 and in FIG. 2.
The MICs for a number of other gram positive and gram negative
organisms has been determined. These bacteria included examples
of the organisms implicated in cases of bacterial sepsis (p. I 157
of the transcript of the Blood Product Advisory Committee meeting
TABLE 1 ______________________________________ MIC .mu.g/ml Ciprofloxacin
______________________________________ 11 Staphylococcus (coagulase
negative) 0.157-0.625 S. epidermidis(QA) 2.5 3 S. aureus 0.625 S.
aureusATCC14504 1.25 4 Micrococcus 1.25-5.0 1 gram positive coccus
1.25 mean 0.94 .mu.g/ml ______________________________________
TABLE 2 ______________________________________ Reference Strains
of Gram Negative Bacilli MIC (.mu.g/ml) Species Strain Ciprofloxacin
______________________________________ Pseudomonas aeruginosa* FDG
5 ATCC27316 0.625 Salmonella typhimurium SL1027 Serratia marcescens
435 0.313 436 437 Klebsiella pneumoniae ATCC8047 ATCC10031 0.156
2CD Serratia marcescens 438 0.078 ______________________________________
*Brain heart infusion medium, all others on MuellerHinton medium
TABLE 3 ______________________________________ Reference Strains
of Gram Positive Cocci MIC (.mu.g/ml) Species Strain Ciprofloxacin*
______________________________________ Enterococcus 101 2.5 Enterococcus
102 1.25 103 104 105 106 107 S epidermidis 1.25 (Q.A. strain) ______________________________________
*In brain heart infusion medium
Platelet Storage Studies with Ciprofloxacin
It has been established that PC inoculated with small numbers of
S. epidermidis will contain massive numbers of bacteria in a few
days. In our own experiments we inoculated PC stored for 1 day with
5 cfu (n=8) or 20 cfu (n=8) of s. epidermidis (Q.A. strain). Massive
growth (>10.sup.7 CFU/ml) was seen within 3 to 4 days after inoculation
(7 of 8 PC and 4 of 8 PC, respectively).
To test the effectiveness of ciprofloxacinin stored platelet concentrates
the following pair study was done. Two units of blood were drawn
from ABO compatible donors. Platelet-enriched plasma (RPR) was prepared
by centrifugation at 2900 RPM for 126 seconds. A PRP pool was made,
sampled for in vitro platelet assays, and then split evenly into
two CLX-7 satellite bags. Platelet concentrates were made by centrifugation
at 3700 RPM for 6 min in an IEC PR6000 floor model centrifuge. All
but 50-55 ml of plasma was removed from the platelet pellet. After
a 90 minute rest period the bags were placed on a horizontal agitator
(70 cycles/min). After 30 minutes on the shaker, when the platelets
were nearly all resuspended, each member of the pair received 10000
cfu of S. epidermidis (n=2) or 10000 cfu of S. aureus ATCC 14504
(n=2). These two species are the ones most often implicated in contaminated
PC. One member of the pair received enough (250 .mu. g/ml) ciprofloxacin
to make the final concentration 5 .mu.g/ml. The bags were returned
to the shaker and stored for 7 days at 22.degree. C. The PRP and
PC at days 1 2 5 and 7 were sampled for the following in vitro
assays: pH, PO.sub.2 the platelet concentration (count), recovery
from hypotonic stress, and the sizing parameters, geometric standard
deviation and the mean volume. Note that the size of the inoculum
was 500 to 10000 times greater than the inoculum needed to get
massive bacterial growth in most PC.
As to be expected there was massive growth by day 2 in those units
not receiving ciprofloxacin (1.4 to 8.0.times.10.sup.7 CFU/ml).
In the 4 PC receiving 5 .mu.g/ml ciprofloxacin, no viable bacteria
were recovered during storage (0 CPU/ml on days 1 2 5 and 7).
The results of the in vitro assays for the PC receiving ciprofloxacin
were averaged and compared with historical data (n=21) in CLX-7
(see FIG. 3). The ciprofloxacin platelet data was comparable to
historical data. The ciprofloxacin PO.sub.2 is lower than historical
because the platelet concentration (count) was unusually high. We
have seen counts as low as 600000/.mu.l to as high as 2600000/.mu.l.
From other studies we have found that high PO.sub.2 affects adversely
the hypotonic stress assay which is consistent with the results
Under blood banking conditions, addition of ciprofloxacin to PC
at low concentrations killed hugh inocula of the two common skin
organisms (S. aureus and S. epidermidis) most often implicated in
contaminated PC. In vitro platelet parameters were not affected
by the presence of ciprofloxacin.
Numerous existing blood bag systems could be adapted to include
the presence or addition of a quinolone carboxylic acid derivative
such as ciprofloxacin. For example U.S. Pat. No. 4484920 shows
a container for mixing a liquid and a solid initially placed in
separate compartments. The blood or blood component would be the
liquid, the quinolone carboxylic acid derivatives could be in a
separate compartment as either a solid, such as a powder or pellet
formulation, or as a liquid.
One method of introducing an appropriate amount of a quinolone
carboxylic acid derivative is to include the quinolone in a satellite
bag which is attached to at least one of the main blood bags. The
quinolone could be in liquid, power or pill formulation. One embodiment
would be to provide an appropriate amount of quinolone, for example
250 .mu.g of ciprofloxacin in a pill formulation. The pill formulation
could be introduced into the blood bag system by activating an opening
and allowing the quinolone to be admixed with the blood or blood
components. The quinolone could be dissolved or flushed from the
satellite pouch by introducing the blood or blood components into
the satellite bag and then allowing the blood or blood components
to be returned to the main blood bag. Alternatively, the appropriate
amount of quinolone or other antibacterial agent would be in a liquid
The introduction of an antibacterial agent into a blood bag storage
or collection system serves to destroy or supress the growth or
reproduction of bacteria. Quinolone carboxylic acid derivatives
have provided the above function without negatively affecting the
viability of the blood or blood components. Other yet to be developed
quinolones are deemed to be within the scope of this invention,
as are other antibacterial agents that provide the desired function.
Given the above disclosure, it is thought variations will occur
to those skilled in the art. Accordingly, it is intended that the
above example should be construed as illustrative and the scope
of the invention should be limited only by the following claims.