Note: Descriptions are shown in the official language in which they were submitted.
STABILIZATION OF LEUKOCYTFS
This invention relates to the stabilization or
preservation of human leukocytes, particularly
granulocytes, and pertains more specifically to the
storage of such cells at temperatures above freezing in
a non-toxic physiologically acceptable medium containing
plasma and gelatin as essential ingredients. After
storage in such a medium, the cells substantially
display most of their original physiological properties
and are of use in the treatment of diseases
characterized by a qualitative or quantitative
deficiency of these cells.
Although there has been an extensive
development of blood banks for collecting human blood
and separating it into several di~fferent components such
as red cells, white cells including neutrophils,
platelets, and serum, the maximum physiological or
therapeutic use of the components can be made only if
they are capable of being stored until needed and/or
transported to the place of need. Certain components
such as red blood cells and serum can be stored for long
periods after freezing under certain conditions, then
reconstituted for use. Limited storage of platelets is
possible, but they lose their ability to function after
at most five days of storage at temperatures above
freezing; and the freezing of platelets for storage,
though feasible (Kotelba-Witkowska et al., Transfusion,
Vol. 22, 121 ff. (1932) is an expensive and little
used technique. Neutrophils, on the other hand, have
been subject to agglutination or clumping after storage
for as little as 48 hours at temperatures above freezing
and so have had to be used within two days of
preparation; storage of neutrophils by freezing has met
with no success.
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It has been proposed in Tullis, Blood, Vol. 6,
772-773 (1951) to employ an isotonic buffer containing
gelatin together with EUTA, ascorbic acid, sodium
acetate and phenol red indicator as a storage medium fo~
leukocytes. Crowley et al., Transfusion, Vol. 14,
574-580 (1974) described decantation of leukocyte-rich
plasma from whole blood drawn in buffer containing 0.4%
gelatin based on the total blood-buffer composition and
stored at 4C with or without the addition of various
supplements but function of the granulocytes declined
markedly after storage for a week. Contreras et al.,
Cryobiology, Vol. 17, 243-251 (1980) described storage
of granulocytes in various plasma-containing storage
media at 4C but unsatisfactory decline in functionality
occurred after 4 days. Contreras et al., Transfusion,
Vol. 18, 46-53 (1978) described s~orage of granulocy~es
in media containing either plasma or acid-modified
gelatin at 4C or 22C with extensive loss of cells
after 3 days. Price et al., Transfusion,
~0 Vol. 25, 238-241 (1985) described the use of
acid-modified fluid gelatin as a red cell sedimenting
agent in the collection of granulocytes but no storage
of the granulocytes occurred prior to testing. Sher et
al., Immunology, Vol. 31, 337-341 (1976) described use
~5 of gelatin as a red cell sedimenting aid in the
collection of granulocytes, but the gelatin was washed
from the granulocytes before suspending them in an
incubation medium with no extended storage.
It has now been found that granulocytes can be
effectively stabilized for storage at temperatures below
25C, preferably below 8C over extensive periods of
time of at least five days or even seven days or more
provided they are suspended in a suitable buffer, one
which is non-toxic and physiologically acceptable,
containing gelatin and human plasma dissolved therein.
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According to one aspect of the present invention there
is provided the method of stabiliziny for storage without freezing
human granulocytes obtained from blood which comprises suspending
them in a non-toxic buffer containing gela~in and plasma in which
the amount of said gelatin is at least 1% by weight of the -total
composition and less than -the amoun~ required to cause said
composition to set to a gel at 40C or higher and the amount of
said plasma is from 25 to 90% by weight of the total composition.
According to a further aspect of the present invention
there is provided a composition comprising granulocytes suspended
in a non-toxic buffer at pH 6.1 to 8.0 containing gelatin and
plasma, the amount of said gelatin is at least 1% by weight of the
total composition and less than the amount required to cause said
composition to set to a gel at 40C to higher and the amount of
said plasma is from 25 to 90% by weight of the total composition.
Aft.er storage, the granulocytes can be ~ransfused without further
processing except for warming to a temperature no hiyher than 40C
to liquefy any gel which is present; or the granulocytes can be
reconstituted for use simply by washing out the gelatin and plasma
with buffer which can be the same as or different from the buffer
used for storage, or by removal of the gelatin-containing buffer
by centrifugation followed by resuspending the granulocy-tes in a
desired buffer or medium.
The buffer employed can be any conventional non-toxic
buffer which provides a pH in the desired range. The pH of the
buffer may vary over a wide range, from about pH 6.1 to about pH
8.5, preferably to about pH 8.0; preferably the p~l is maintained
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near the neutral point, e.g. about 6.5 to about 7.5 in order to
optimally preserve function. For best results, the granulocytes
and platelets in plasma- and gelating-containing buffer should be
stored at low temperature, e.g. below 8C, althouyh they may be
stored at higher temperatures up to ahout 25 for at least 12
hours with satisfactory results.
Gelatin from any of the usual commercial sources can be
used in the practice of the present invention. The amount of
~elatin may vary over a wide range, depending in part on the
length of storage desired. As little as 1.0~ by weight based on
the total weiyht of the total buffer-plasma composition is
effective for short storage; there is no critical upper limit on
the amount used, except that it must be low enough so that the
composition is a liquid, rather than a gel, a~ a temperature no
higher than 40C in order to facilitate removal of the gelatin
after storage.
The concentration or number of the granulocytes in the
buffer may vary over a limited range, from 107 to 109 per ml,
preferably from 1 x 107 to 2 x 108, more preferably from 1 x 108
~0 to 5 x 108 per ml.
The plasma employed can be normal human plasma or
autologous plasma. The amount of plasma may vary from 25% by
weight of the total composition to as much as 90% by weight.
A local anesthetic such as lidocaine is an optional
additive which aids in maintaining the cell count at a high level.
Other local anesthetics which can be used include procaine,
mepivacaine, bupivacine, and the like.
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Tests have shown -that granulocytes which have be~n
stored for five days or even seven days or more in accordance with
the present invention, then reconstituted by 25-fold dilution of
the gelatin and plasma, largely retain all of the desired
physiological properties and are substantially free from
agglutination or clumping. In particular, they are capable of
ingesting and killing opsonized microorganisms.
The following specific examples are intended to
illustrate more fully the nature of the present invention without
acting as a limitation upon its scope.
In each of the following examples, granulocytes were
prepared from freshly drawn human blood which had been
anticoagulated by the addition of 15-18% by volume of an acidic
and hypertonic buffer, an aqueous solution containing sodium
citrate and citrlc acid (0.38 M in citrate and citric acid and 20
mg/ml of glucose at pH 4.8) (ACD). The granulocytes were
separated by dextran sedimentation and centrifugation as described
in Curnutte et al., J.Clin. Invest., Vol. 53, pp. 1662-1672
(1974). They were then washed in Dulbecco's phosphate buffered
saline without calcium or magnesium (PBS) and without removing red
cells by hypotonic lysis.
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The granulocytes were then suspended in tne
neutral histidine buffer used as the storage medium:
0.1 M sodium citrate, 0.05 M histidine, 0.1 M glucose,
0.05 M sodium pyruvate, and 1 ~M lidocaine adjusted to
pH 7.4 (HCP).
Autologous human plasma was drawn in the same
HCP buffer, 10 ml of blood being mixed with 1.4 ml
buffer and spun for 10 minutes successively at 1500 and
2000 rpm respectively with separation of the plasma
fraction after each centrifugation.
The following compositions were then prepared,
the proportions of gelatin and plasma being expressed as
a percentage by weight of the total:
lSComposition Gelatin Plasma Lidocaine
No. % % (~ M)
1 2 44 o.~
2 1 44 0-5
3 0.5 0 0.5
4 0 0 0.5
44
6 0.5 44 0
7 1 0 0
8 2 0 0
~5 In each case the concentration of granulocytes was 2 x
per ml. and the pH was 7.4 at room temperature.
Five specimens of each of the compositions were
stored in a refrigerator at 4C.
After storage for 7 days at 4C, each specimen
was allowed to warm to room temperature; those specimens
which were gels at 4C liquified upon warming. The
specimens were diluted tenfold with Dulbeccols
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phosphate-buffered saline without calcium and
magnesium. Granulocyte function was then evaluated.
Survival of granulocytes was measured by cell count,
expressed as a percentage of the initial cell count
before storage. Viability was determined by the
percentage of cells which excluded trypan blue when
suspended at 0.4% by weight in PBS buffer without
calcium and magnesium. Capability of bacterial killing
was measured with respect to S. aureus using
substantially the same procedure as described by Babior
et al. in Leukocyte Function, Cline ed., pp. 1-38 (NY
1981).
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i`he results were as follows (average of five
specimens):
Composition Cell Count Viability Bacterial
No.~ initial % killinq %
1 109 74 98
2 62 59 9S
3 18 13 74
4 31 30 69
S 32 18 99
6 22 27 96
7 58 60 57
8 71 73 73
From the foregoing result~s it is clear that
cell counts, viability, and bacterial killing capacity
~ are all preserved to a much greater extent in the
compositions containing gelatin and plasma in
combination, whereas two or even all three functions are
greatly reduced when one or both of these ingredients
are omitted.
What is claimed is:
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