A multiple blood bag system is disclosed having at least two blood
bags and conduit means providing sealed flow communication between
the bags and a filtering means integrally disposed between two of
the bags for removing platelets and white cells from a red cell
concentrate in the blood bag system. In the method of the invention
a red cell concentrate is provided in one of the blood bags of the
above system and an additive solution is mixed therewith. The mixture
of the red cell concentrate and additive solution is passed through
the filtering means from one of the bags to another.
1. In a multiple blood bag system comprising a primary bag and
at least two satellite bags each connected by conduit means to the
primary bag and providing sealed flow communication between the
bags, the improvements which comprise one of the satellite bags
containing an additive solution for mixing with a red cell concentrate,
and a filtering means integrally disposed between the primary bag
and the satellite bag containing the additive solution such that
the additive solution of the satellite bag may be passed through
the filter to the primary bag and red cells in the primary blood
bag may be passed through said filtering means to the satellite
2. The multiple blood bag system of claim 1 wherein the primary
bag is integrally fitted with a needle and tubing for collecting
blood from a donor.
3. The multiple blood bag system of claim 1 wherein the filtering
means comprises a housing containing a filtering medium.
4. The multiple blood bag system of claim 3 wherein the filtering
medium is selected from the group consisting of cotton wool, cellulose
acetate, and synthetic fibers.
5. The multiple blood bag system of claim 1 wherein the filtering
means removes platelets and white cells thus preventing formation
of microaggregates in red cell concentrates.
6. The multiple blood bag system of claim 1 wherein the additive
solution is a red cell storage solution.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to and has among its objects novel apparatus
and methods for providing packed red blood cells (erythrocytes)
which are substantially free of platelets and contain low levels
of white cells and have a storage life of up to about 35 days. Further
objects of the invention will be evident from the following description
wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
It is desirable, for patients who require frequent blood transfusions,
to remove white cells and platelets from donor blood prior to transfusion.
Febrile reactions, which are usually attributable to transfused
HLA antigens on white cells and platelets present in the blood may
occur in 3.6% of patients infused. Such side effects are usually
more frequent and severe in patients who receive multiple transfusions.
The removal of HLA antigen may be accomplished in a number of ways.
Perhaps the most effective means, but unfortunately the most costly,
involves the reconstitution of frozen blood.
A less expensive approach is to wash blood with saline in a number
of washes. However, this saline wash approach is time consuming
not only in the processing of batches but also in continuous operation.
Furthermore, significant loss of red cells occurs and less predictable
removal of white cells and platelets is obtained.
Kikugawa et al in Vox Sang., Vol. 34 281-290 (1975) describe commercial
cotton wool filters for filtering blood to remove the above HLA
antigen. These filters are, however, expensive and cumbersome to
Diepenhorst et al in Vox Sang., Vol. 23 308-320 (1972) and Vol.
29 15-22 (1975) disclose cotton wool filtration of blood under
pressure. This method, while efficient, requires a special apparatus
that is expensive.
All of the above techniques require that the treated blood be infused
within 24 hours of treatment in order to avoid the potential risk
of infection. Prolonged shelf life of blood so treated is not possible.
SUMMARY OF THE INVENTION
The invention described herein provides for apparatus and methods
for preparing red blood cells which are substantially free of platelets
and contain low levels of white cells (thus preventing formation
of microaggregates) and have a storage life of at least about 21
days, preferably up to about 35 days, depending on the nature of
the anticoagulant and red cell storage solution employed.
The apparatus of the invention comprises a multiple closed blood
bag system having at least two blood bags and conduit means providing
sealed flow communication between the bags. A filtering means is
integrally disposed between two of the blood bags.
In the method of the invention a red cell concentrate is provided
in a blood bag of a multiple blood bag system comprising at least
two blood bags and conduit means providing sealed flow communication
between said bags and a filtering means integrally disposed between
two of the bags. The red cell concentrate is mixed with an additive
solution and the mixture is passed through the filtering means from
one of the bags to the other. The filtering means is designed to
remove platelets and white cells, and thus prevent formation of
microaggregates in the red cell concentrate.
The primary advantage of the present invention is its simplicity
and its effectiveness. The apparatus of the invention is easy and
inexpensive to use. A further advantage of the invention is that
the red cells prepared as above have an extended storage life of
at least about 21 days or more. Above all, the filtered red blood
cells are essentially free of platelets and have very low levels
of white cells and microaggregates, thus reducing the incidence
of febrile reactions or HLA sensitization in the infusion of the
treated red cells.
The blood bag system having an integral filter assures that sterility
will not be breached during the filtering operation. Prolonged shelf
life is assured, and there is no added risk of infection.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a three-dimensional depiction of an apparatus in accordance
with the present invention.
FIG. 2 is a three-dimensional depiction of a filtering means in
accordance with the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In a preferred embodiment the multiple blood bag system of the
invention comprises at least three bags, a primary bag and at least
two satellite bags, connected by conduit means providing sealed
flow communication between the bags and a filtering means integrally
disposed between two of the bags. Red blood cells in the multiple
blood bag system may be passed from one of the bags to another through
the filtering means comprising a housing containing a filtering
medium which removes platelets and white cells and provides extended
storage life of the red cells when the red cell concentrate is mixed
with an additive solution, e.g., a red cell storage solution, prior
to filtration by introducing the additive solution into the red
In the method of the invention whole blood is introduced, e.g.,
by collection from a donor, into the primary bag of the above system.
After centrifugation, plasma is expressed into one of the satellite
bags which does not have a filtering means disposed between it and
the primary bag. Next, the additive solution in the satellite bag
that is separated from the primary bag by the filtering means is
passed through the filtering means into the donor bag wherein it
is mixed with the red cells. The mixture of red cells and solution
is then passed back through the filtering means into the satellite
bag in which they may be stored for extended periods. The so-treated
red cells are substantially free of platelets (i.e., containing
less than 10% of the original platelets) and of white cells (i.e.,
containing less than 10% of the original white cells); also microaggregate
formation is virtually eliminated.
The apparatus and method of the invention will next be described
in detail with reference to FIGS. 1 and 2.
Blood bag system 10 includes primary or donor bag 12 connected
to satellite bags 14 and 16 by means of flexible tubing or conduit
means 18 which provides sealed flow communication between 12 and
14 and 16. Bag 12 is adapted for receiving blood from a donor and
includes blood collection tube 20 and donor needle 22. Satellite
bag 26 is connected to bag 14 by means of flexible tubing 18.
Fluid flow through tubing 18 is controlled by conventional valving
means such as snap-off plugs, removable plugs, or slide clamps.
Conventional access ports 28 30 32 and 34 are found on bags 12
14 16 and 26 respectively, which may also contain other design
features known in the art.
The blood bags of system 10 may be of conventional construction
being made of a plastic material that is blood compatible, flexible,
translucent, and sterilizable. The plastic may be a polyvinylchloride,
polyester, polyolefin, polyurethane, and so forth and may include
blends of the above materials. Flexible tubing 20 and 18 may be
made of a plastic material that is the same as or different from
the plastic material of the blood bags.
Filtering means 26 is integrally disposed between bags 12 and 16
and is attached at its ends to tubing 18. Filtering means 26 includes
a housing 36 made of rigid polyvinylchloride or the like and tubing
fitments 38. Filtering means 26 is filled with a filtration medium
40 such as cotton wool or cellulose acetate or other synthetic fibers
such as polyester, polyamides, and the like. Preferred filtration
medium for purposes of this invention is either cotton wool as prepared
by the method of Diepenhorst et al referred to above (incorporated
herein by reference) or cellulose acetate. The amount of filtration
medium depends upon the amount of red cells to be filtered. Usually
about 20-50 grams of filtration medium are employed per 200-250
ml of red cell concentrate.
Bag 16 contains an additive solution for mixing with the red cells
to be filtered to prolong the storage life of the red cells. This
additive solution may be, for example, a conventional red cell storage
solution such as that described in Ginzburg et al, Bibl. Haemotol.,
1971 No. 3 Pt. 2 217-220; Wood et al, Blood, Vol. 42 No. 1
1973 17-25; Beutler, "The Red Cell in Vitro", Grum and
Stratton, New York, N.Y., 1974 p. 201; Lovric et al, Medical Journal
of Australia, Vol. 2 183-186 1977; U.S. Pat. No. 4267269; in
an amount of about 50-100 ml per 200-250 ml of red cell concentrate.
In use, blood is collected into bag 12 through donor tube 20 under
conventional conditions. Donor bag 12 may contain an anticoagulant
such as Adenine-Citrate-Dextrose (ACD), Citrate-Phosphate-Dextrose
(CPD), Citrate Phosphate-277 millimoles Dextrose (CP2D), CPD plus
adenine, or other conventional anticoagulant, with which the collected
blood mixes. The collected blood may then be processed directly
or stored usually at about 4.degree.-6.degree. C. At processing,
bag system 10 is centrifuged as is customary in the art causing
the red cells in the blood in bag 12 to settle at the bottom of
the bag. Blood plasma is expressed by conventional techniques from
12 into 14 from which fresh plasma and a platelet concentrate, for
example, may be obtained in bags 14 and 26 respectively. Bag system
10 may optionally be equipped with other satellite bags into which
other blood components may be expressed or processed as necessary
The additive solution from bag 16 is drained through filter 26
into donor bag 12 now containing the red blood cell concentrate.
The red cells are back-filtered, preferably by gravity, through
filter 26 into bag 16. This may be accomplished, conveniently in
the refrigerator or coolroom, at a temperature of about 4.degree.-6.degree.
C., e.g., overnight or for the period of time necessary to filter
all of the red blood concentrate, usually about 2-4 hours or more.
A blood bag system with only two blood bags in sealed flow communication
and an integral filtering means disposed between the bags is within
the purview of the invention. However, in use such a system would
require connection of a third receptacle to receive plasma separated
from the red cell concentrate. Such connection may be made, for
example, at one of the ports of the donor bag by means, for example,
of a sterile connector having a third bag attached thereto for receiving
The invention is demonstrated further by the following illustrative
For experimental purposes 59 units of donor blood (425-450 ml)
were collected into blood bags containing 63 ml of citrate phosphate
and 277 mM dextrose anticoagulant, specially designed for the purpose
by Tuta Laboratories, Lane Cove, Sydney, Australia. (For purposes
of carrying out these experiments the donor bag was part of a blood
bag system of the type exhibited in FIG. 1 but containing an additional
satellite bag integrally attached by means of flexible tubing to
the donor bag.) Following centrifugation at 4000 g for 10 minutes,
the plasma was removed. The residual packed red cells were split
into two halves. One half was suspended in a satellite bag containing
50 ml of an aqueous additive solution of disodium hydrogen phosphate
(25 mM), adenine (0.5 mM), sodium chloride (123 mM), dextrose (40
mM), trisodium citrate (15 mM), and citric acid (9.75 mM). Control
data were obtained from this portion of the packed red cells.
The test data were obtained from the other half of the packed red
blood cells; the same additive solution was also contained in a
satellite bag separated from the donor bag by a filter integral
with the tubing connecting the donor and satellite bag. This 50-ml
volume additive solution was passed through the filter into the
donor bag containing the remaining half of the residual packed red
cells. The filter medium comprised cotton wool (20 g) prepared by
the method of Diepenhorst et al mentioned above. Next, the red cell
concentrate in the donor bag was filtered overnight by gravity at
4.degree.-6.degree. C. through the filter and into the satellite
bag integrally connected therewith.
Autologous erythrocyte survival studies (Dacie et al, "Practical
Haemotology", 5th ed., Churchill Levingston, Edinburgh, 1975)
were assessed in 10 volunteers following informed consent, and were
determined either at 28 or 35 days of storage. Oxygen dissociation
curves were drawn with the Hem-O-Scan instrument (Aminco, Silver
Springs, Md.). The P.sub.50 values were determined by first removing
the supernatant after centrifugation. The packed red cells were
then adjusted to a hematocrit of 40% and pH 7.4 with a reagent mixture
containing AB plasma and Tris-HCl buffer (pH 9.0) and then tested
immediately. Adenosine triphosphate (ATP), 23-diphosoglycerate
(23-DPG) supernatant hemoglobin, pH, potassium and methylene blue
uptakes were tested as described in Lovric et al, Vox Sang., Vol.
33 346-352 1977. Erythrocyte deformability was measured with the
5-.mu.m polycarbonate filtration method (Ried et al, J. Clin. Path.,
Vol. 29 853-855 1976) using a negative pressure of 130 mm Hg.
Microaggregate counts were done on aliquots kept between 4.degree.
and 6.degree. C. until just prior to counting using a ZF Coulter
Counter with a 200-.mu.m orifice coupled to the Coulter Channelyzer
(Coulter Electronics, Hialeah, Fl.). The packed red cells were first
resuspended by gentle mixing, and a 1/100 dilution made in isotonic
saline (Isoton, Coulter Electronics). Bubble formation could be
avoided by first aspirating the sample into the Coulter diluter,
and subsequent release near the bottom of the vessel already containing
the diluent. Next, erythrocytes were lysed by adding 50 .mu.l of
10% saponin (Fisher Scientific, Fairlawn, N.Y.) and a stop watch
started. It is essential that the saponin be added slowly, the container
inverted slowly twice only and without shaking, lest bubble formation
preclude reproducible counts. Exactly 65 sec. after the addition
of saponin the count is commenced and the mean of duplicates recorded
for each of the channels. The background count values were subtracted
from blanks not containing blood, but handled identically. The coefficient
of variation with this procedure is less than 5%.