CA2004043C - Stabilization of leukocytes - Google Patents

Stabilization of leukocytes

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Publication number
CA2004043C
CA2004043C CA002004043A CA2004043A CA2004043C CA 2004043 C CA2004043 C CA 2004043C CA 002004043 A CA002004043 A CA 002004043A CA 2004043 A CA2004043 A CA 2004043A CA 2004043 C CA2004043 C CA 2004043C
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Canada
Prior art keywords
leukocytes
buffer
concentration
bases
plasma
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CA002004043A
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French (fr)
Other versions
CA2004043A1 (en
Inventor
Bernard M. Babior
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Scripps Research Institute
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Scripps Clinic and Research Foundation
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Application filed by Scripps Clinic and Research Foundation filed Critical Scripps Clinic and Research Foundation
Publication of CA2004043A1 publication Critical patent/CA2004043A1/en
Application granted granted Critical
Publication of CA2004043C publication Critical patent/CA2004043C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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Classifications

    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02Preservation of living parts
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02Preservation of living parts
    • A01N1/0205Chemical aspects
    • A01N1/0231Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes

Abstract

Preservation of leukocytes by suspending in storage medium containing modified fluid gelatin, plasma, and a non-toxic buffer. Preferred additives include heterocyclic bases which occur in nucleic acids, or nucleosides or nucleotides containing the same.

Description

200~0~3 STABILIZATION OF LEUKOCYTES
This invention relates to the storage of human white cells or leukocytes and pertains more specifically to a composition containing human plasma, modified fluid gelatin, and non-toxic buffer, into which leukocytes can be dispersed and stably maintained during storage at low temperature for periods of at least 24 hours, usually at 48 hours, and even up to two weeks or more. Improved results can be achieved by including also in the l0 composition one or more of a) heterocyclic bases (purines or pyrimidines) which occur in nucleic acids; b) nucleosides containing said bases; or c) nucleotides containing said bases. Among suitable heterocyclic bases are adenine, cytosine, guanine, thymine and uracil.
While collection and fractionation of human blood and use of its components for transfusion is widely practiced, use of certain components such as granulocytes or polymorphonuclear leukocytes has been severely limited by the lack of stability of these components during storage, even at 4°C. This lack of stability is manifested primarily by agglutination or clumping of the cells and also involves acceleration of the cell death rate and reduction of bactericidal capacity of the cells.
As a practical matter, these difficulties have greatly restricted the use of such components, since distribution and supply of blood components to points of need require that the components be stored for substantial periods of time up to two weeks or even more.
Moreover, additional storage time would allow additional tests to be carried out such as HLA typing or cross-matching or tests for the presence of cytomegalovirus and other pathogens, thus optimizing benefits of the transfusion procedure.
It has hitherto been proposed to employ plasma and gelatin in a buffer for storage of granulocytes as described in Babior et al. U.S. Patent 4,639,373.
It has now been found that substantially longer storage and less degradation of leukocytes during storage at temperatures below 25°C, preferably below 8°C but above freezing, can be achieved by employing in combination plasma, modified fluid gelatin, and a physiologically acceptable non-toxic buffer. Modified fluid gelatin has been used clinically as a plasma substitute or expander and is widely available under a variety of trade-marks, such as PlasmaGel, Haemaccel, Leukogel, Gelofusine, etc. In general, these materials are partly hydrolyzed gelatins which have an average molecular weight from 15,000-40,000 daltons and which form aqueous solutions having a viscosity less than that of a gelatin solution of the same concentration. In some cases they are succinylated, or they are reacted to form e.g. urea linkages, or cross-linked. The commercially available materials usually are in the form of a solution of the modified gelatin in a buffer together with various salts and other ingredients. The term ~~modified fluid gelatin" as used herein refers to the partly hydrolyzed gelatin component itself whether or not further reacted.
In some cases the commercially available solutions can be used, but preferably the partly hydrolyzed gelatin component is purified by separation from the remaining ingredients of the commercial product.
Any conventional buffer solution can be used in the compositions of the present invention such as any of the buffers commonly used in connection with storage of blood or its components at a pH from 4 to 7.5, e.g., Citrate Phosphate Dextrose (CPD) which consists of 2.06 grams citric acid, 16.6 grams sodium citrate, 16.1 grams dextrose and 1.40 gram monobasic sodium phosphate 3 ~, Q~ ~ ~ ~ 4~ ~
dissolved in deionized water to make 630 ml; or Acid Citrate Dextrose (ACD) which consists of 14.75 grams citric acid, 29.0 grams sodium citrate, and 20.0 grams dextrose dissolved in deionized water to make 500 ml. The ACD buffer is preferred because compositions made with it maintain better the bacterial killing activity of the leukocytes during storage.
The heterocyclic base component (free base, nucleoside, or nucleotide), when present, may be present in very small concentrations, as little as about 0.001 mM being l0 effective to enhance stability of leukocytes dispersed in the compositions, or preferably up to about 5 mM, but the preferred concentration range is from 0.5 to 3 mM in the composition.
The plasma present in the composition may be normal human plasma, either autologous or heterologous with the leukocytes, preferably autologous. The amount of plasma present in the composition must be at least 25% by weight based on the total composition exclusive of the leukocytes, while the preferred amount is from 40 to 50% by weight.
20 Larger amounts of plasma up to about 75% by weight can be used.
The amount of modified fluid gelatin in the composition may vary considerably, from 4% by weight based on the total composition, exclusive of leukocytes, up to the amount which causes the composition to set to a gel at 40oC, preferably 6-25% by weight. The amounts required for optimum results vary depending upon the source of the modified fluid J
gelatin but can readily be determined in any given case by a simple test.
The leukocytes can be dispersed in the composition in amounts varying from 10~ to 109 cells per ml, preferably from 1 x 108 to 5 x 108 cells per ml.
During storage of the leukocyte-containing composition, it is preferably maintained at a temperature below 8°C although higher temperatures up to 25°C can be tolerated for relatively short times. For optimum storage life, storage temperature should be maintained at 4oC or lower, but not below freezing temperature.
After storage the composition containing the leukocytes can be transfused after warming without further processing; or if desired the leukocytes can be separated from the composition by washing or centrifuging in order to permit dispersion of the cells in any other desired medium.
It is contemplated that the present invention may be practiced by supplying to blood banks, laboratories or other entities a stock solution suitable for mixing with a suspension of leukocytes in plasma, free from red cells, with directions for mixing the stock solution with the suspension in suitable proportions. Such a stock solution comprises a water solution containing an amount of modified fluid gelatin from 8% by weight up to the amount causing the solution to gel at room temperature, preferably 20 to 40% by weight, and a member of the group consisting of a) the heterocyclic bases which occur in nucleic acids, b) nucleosides containing said 4a bases, c) nucleotides containing said bases, and d) mixtures of a), b) and c), said member being present at a concentration from 0.5 micromolar to 20, preferably to 10 millimolar, preferably from 0.8 to 8 millimolar. The member preferably is adenosine or guanosine. The stock solution may also include a non-toxic buffer at a pH of 4-7.5, preferably a citrate-based buffer such as ACD or CPD at a concentration of 100-500 milliosmoles per liter.
The following specific examples are intended to illustrate the invention without acting as a limitation upon its scope.
In each case of the following examples the plasma employed was prepared by drawing 20 ml of whole blood into a 30 ml syringe preloaded with either 4 ml ACD
buffer or with 2.8 ml CPD buffer, then spinning for ten minutes at 2,000 rpm in a centrifuge at 4°C. The supernatant was separated and used as plasma.
The leukocytes (neutrophils) used in these examples were prepared from whole human blood by drawing a 30 ml specimen of blood into a 60 ml syringe pre-loaded with 6 ml ACD buffer; to the mixture was added 20 ml of 6% dextran (GENTRAN*70, Pharmacia) in normal saline (0.9%), and after standing, the straw colored supernatant was separated and spun ten minutes at 800 rpm in a centrifuge at 4°C; the resulting pellet was resuspended in 3.5 ml Dulbecco~s Phosphate Buffered Saline without calcium chloride and without magnesium chloride (DPBS--).
Four such resuspensions were combined in a centrifuge tube, vortexed, then spun 4 minutes at 1200 rpm at 4°C in a centrifuge and the supernatant discarded. The red cells were lysed by directing onto the pellet a 6 ml stream of ice cold deionized water, vortexing for 30 seconds, then quickly adding to the suspension 2 ml of 0.6 M potassium chloride solution and 3.5 ml DPBS--. The tube was then spun 4 minutes at 800 rpm at 4°C, the supernatant discarded, and the pellet washed gently with 3.5 ml DPBS--. The lysis, washing and spinning was repeated, after which the pellet was resuspended in 3.5 ml DPBS--, pooled with a second pellet prepared in the same manner, and DPBS-- added to a total volume of 20 ml;
the suspension was underlaid with 10 ml Ficoll Hypaque;
and the tube was spun 20 minutes at 1200 rpm. After removing and discarding the liquid layers, the pellet remaining was resuspended in 2.5 ml of autologous plasma prepared as described above, and the concentration was adjusted to 2 x 108 cells per ml, twice the concentration desired in the finished storage composition of the example.
*Trade-mark The modified fluid gelatin of each example was prepared by purifying commercially available material supplied under the specified trade name by dialyzing exhaustively against water, then lyophilizing. It was then dissolved, for those examples using ACD as the buffer, in the buffer at a concentration such that, after mixing with plasma, the resultant storage solution would have the desired concentration, then mixed with the desired volume of leukocyte-containing plasma prepared as described above and containing the corresponding buffer to form the specimen of storage composition. Where desired, nucleoside bases, nucleosides, and nucleotides were dissolved in the modified fluid gelatin solution before mixing with plasma.
In making storage compositions containing CPD
buffer with no heterocyclic base component, the buffer was diluted at 7 parts CPD to 3 parts water before dissolving the modified fluid gelatin in it. When heterocyclic base component was dissolved in addition to the modified fluid gelatin, the buffer was diluted at 6 parts CPD to 4 parts water before dissolution, and both the modified fluid gelatin and heterocyclic base component were dissolved in the diluted buffer to provide the concentration desired in the ultimate storage composition after mixing with plasma.
Storage compositions having the components shown in Tables 1-3 below were prepared by the foregoing procedures, the concentration of modified fluid gelatin and the approximate concentration of plasma in each case being expressed as percentage by weight of the total composition excluding leukocytes.
After storage for seven days at 4°C, the cell recovery (cell count expressed as a percent of initial cell count), cell viability, and bacterial killing power of the cells were determined. These values were zoo~o~~
_,_ determined by bringing the storage sample up to room temperature, spinning 5 minutes at 800 rpm, discarding the supernatant, and resuspending the pellet in 0.6 ml DPBS--; 10 ~L of the suspension was diluted in 20 ml of Isoton and counted to determine recovery.
To determine viability, a drop of the resuspension was mixed with an equal volume of 0.2% Trypan Blue in normal saline and incubated 5 minutes at room temperature, after which 100 cells were counted l0 microscopically (blue stained cells were non-viable).
The remainder of the resuspension was used for bacterial killing assay.
The bacterial killing assay was conducted using a clinical isolate of _S. aureus from a patient. The isolate was suspended in Hank's Balanced Salt Solution at a concentration of 1 x 108 organisms/ml. Autologous serum for use in the assay was prepared by allowing a 5 ml specimen of whole blood to clot for an hour in a culture tube; the clot was loosened, and the tube spun for 10 minutes at 3000 rpm at room temperature. The supernatant was drawn off and used as the serum.
Duplicate assays were conducted for each example by first diluting the resuspension described in the preceding paragraph to a concentration of 2 x 10~ cells per ml, then mixing with serum and buffer in the following proportions to form a test specimen:
200 _S. aureus suspension at 1 x 108 bacteria/ml 50 Autologous serum 200 test sample at 2 x 10~ cells/ml in DPBS--Blanks (controls) were run containing DPBS-- with no neutrophils.
A 1001 aliquot of each test specimen was diluted immediately 1:10 with DPBS-- to facilitate counting, and the remainder was incubated for 2 hours at 37°C with gentle but thorough agitation. At the end of the ~Q~4C~43 _8_ incubation, an additional 100,1 aliquot of each specimen was diluted 1:10 with DPBS--. Each of the two dilutions (and two corresponding dilutions of the blanks) was subjected to the following procedure to determine the proportion of surviving bacteria. A 101 aliquot of each dilution was mixed with 4 ml sterile deionized water, sonicated 2-3 seconds to break up clumps, and allowed to stand 5 minutes at room temperature. Top agar was prepared by mixing with Nutrient Broth 0.65% by weight of agar, autoclaving to dissolve, then maintaining at 50°C.
A 10 microliter portion of the sonicated solution was added to 5 ml of top agar at 50°C and poured over a 1.5%
nutrient agar plate and allowed to solidify at room temperature, after which the plate was inverted and incubated overnight at 37°C. The colonies were then counted over a light box. Bacterial killing was calculated as follows:
Percent = 100-(surviving bacteria in test specimen) (100 Kill surviving bacteria in blank Normal percent kill for fresh leukocytes before storage is >90%.
The results are shown in Tables 1, 2 and 3 below:

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n m 9 Additional storage samples were prepared having compositions as shown in Table 4 below using the same procedures as described above with ACD as the buffer and leukocytes in each case at a concentration of 1 x 108 cells/ml.
These storage compositions were held for two weeks instead of 7 days at 4°C. After storage, leukocyte retrieval was facilitated by diluting the storage composition with an equal volume of phosphate-buffered saline, which reduced viscosity and facilitated accurate pipetting. Recovery and viability were measured immediately after dilution instead of after isolation and resuspension of the leukocytes, thus improving reliability of these measurements. In addition, the bacteria used in the bacteria killing assay were _S.
aureus strain 502A from the American Type Culture Collection. The results were as follows:

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i 20~~40~3 Additional storage samples were prepared as described above having the composition:
Percent Plasmagel 12%
Plasma 45%
containing ACD as the buffer and containing various concentrations of heterocyclic bases or of nucleosides, as shown in Table 5 below. After one week storage at 4°C
the leukocyte recovery, viability, and bacterial kill were determined as described above with the results shown in Table 5.

2Q~4~D~3 Concentration)leukocyte Bacterial Heterocvclicnrl Recovery Viability KiltX
Base X X

SE SE SE

Adenine 2.0 67.3 0.5 75.3 4.0 56.322.6 Adenine 0.2 72.3 8.2 72.7 3.7 37.030.4 Cytosine 1.0 68.7 11.5 90.7 5.8 75.318.7 Uracit 0.2 68.3 5.2 82.0 4.5 70.615.3 R i bosY
l rxx l eos i de Cytidine 2.0 65.0 7.5 81.7 6.5 53.829.9 Cytidine 0.2 76.3 15.3 81.0 7.0 47.735.4 Guanosine 1.0 69.0 5.7 89.3 6.1 89.32.8 Uridine 1.0 66.3 13.1 86.0 14.1 83.26.5 204~404~

Generally similar results can be obtained with various other free bases, nucleosides or nucleotides.
What is claimed is:

Claims (13)

1. The method of stabilizing for storage leukocytes obtained from human blood which comprises mixing a suspension of said leukocytes in plasma with modified fluid gelatin and with a non-toxic buffer.
2. The method of stabilizing for storage leukocytes obtained from human blood which comprises suspending said leukocytes in plasma containing modified fluid gelatin and a non-toxic buffer.
3. The method as claimed in claim 1 wherein said mixture includes one or more of a) the heterocyclic bases which occur in nucleic acids; b) nucleosides containing said bases; or c) nucleotides containing said bases.
4. The method as claimed in any of claims 1, 2 or 3 including in addition the step of storing said mixture for at least 24 hours at a temperature of 25°C or below.
5. A composition for maintaining the viability and bactericidal capacity of human leukocytes during storage comprising 25 to 75% by weight of human plasma, an amount of modified fluid gelatin of from 4% by weight up to the amount causing the composition to gel at 40°C, a member selected from the group consisting of a) the heterocyclic bases which occur in nucleic acids, b) nucleosides containing said bases and c) nucleotides containing said bases at a concentration from 0.001 to 5mM, human leukocytes at a concentration from 10 7 to 9 cells per ml, and the balance a non-toxic aqueous buffer.
6. A composition for maintaining the viability and bactericidal capacity of human leukocytes during storage comprising 40-50 percent by weight of human plasma, 6-25 percent by weight modified fluid gelatin, a nucleoside selected from adenosine or guanosine at a concentration from 0.001 to 5mM, human leukocytes at a concentration from 10 7 to 10 9 cells per ml, and the balance a non-toxic aqueous buffer.
7. A composition as claimed in claim 5 in which said member is adenosine.
8. A stock solution adapted to be mixed with a suspension of human leukocytes in plasma to maintain the viability and bactericidal capacity of said leukocytes during storage, said solution comprising a water solution containing an amount of modified fluid gelatin from 8% by weight up to the amount causing the solution to gel at room temperature, and a member selected from the group consisting of a) the heterocyclic bases which occur in nucleic acids, b) nucleosides containing said bases, c) nucleotides containing 18a said bases, and d) mixtures of a), b) and c), said member being present at a concentration from 0.5 micromolar to 20 millimolar.
9. A stock solution as claimed in claim 8 comprising in addition a non-toxic buffer at pH 4 to 7.5.
10. A stock solution as claimed in claim 9 in which the amount of said modified fluid gelatin is from 20 to 40% by weight.
11. A stock solution as claimed in claim 10 in which the concentration of said member is from 0.8 to 8 millimolar.
12. A stock solution as claimed in claim 10 in which said buffer is a citrate based buffer at a concentration of 100 to 500 milliosmoles per liter.
13. A stock solution as claimed in claim 11 in which said member is adenosine or guanosine and said buffer is a citrate based buffer at a concentration of 100 to 500 milliosmoles per liter.
CA002004043A 1988-11-29 1989-11-28 Stabilization of leukocytes Expired - Fee Related CA2004043C (en)

Applications Claiming Priority (2)

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US07/277,256 US4923797A (en) 1988-11-29 1988-11-29 Stabilization of leukocytes
US07/277,256 1988-11-29

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Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4923797A (en) * 1988-11-29 1990-05-08 President & Fellows Of Harvard College Stabilization of leukocytes
AU650678B2 (en) * 1990-08-10 1994-06-30 Analytical Control Systems, Inc. Stabilization (using gelatin from a cold water fish) of enzymes in diagnostic controls
EP0647267B1 (en) * 1992-06-25 2000-08-30 SCHEER, David Stem cell storage
GB2314843A (en) * 1993-07-05 1998-01-14 North Gen Hospital Nhs Trust Whole blood comprising a stabilised leucocyte preparation
US6415201B1 (en) * 1998-04-08 2002-07-02 Andre Lefesvre Process and system for management of batches of components of the haematopoietic system for deferred use
GB0031065D0 (en) * 2000-12-20 2001-01-31 Univ Cardiff Preservation of cells
WO2010003037A2 (en) * 2008-07-03 2010-01-07 Sierra Molecular Corporation Compositions, systems, and methods for stabilization of a cell and/or macromolecule
EP3662750A1 (en) 2011-04-07 2020-06-10 Fenwal, Inc. Automated methods and systems for providing platelet concentrates with reduced residual plasma volumes and storage media for such platelet concentrates
US9089479B2 (en) 2011-05-03 2015-07-28 Fenwal, Inc. Medium, solutions and methods for the washing, culturing and storage of white blood cells

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Publication number Priority date Publication date Assignee Title
US3874384A (en) * 1971-11-01 1975-04-01 American Hospital Supply Corp Improved blood storage unit and method of storing blood
JPS56139419A (en) * 1980-03-31 1981-10-30 Kuraray Co Ltd Erythrocytic preservative and erythrocytic pharmaceutical for preservation
US4558032A (en) * 1981-12-31 1985-12-10 Neomed Inc. Synthetic whole blood substitute and a method of making the same
US4539204A (en) * 1982-10-29 1985-09-03 Neomed Inc. Gelatin based synthetic blood and a method of making the same
US4596788A (en) * 1983-02-07 1986-06-24 Neomed, Inc. Gelatin and lecithin based synthetic whole blood and a method of making the same
US4471051A (en) * 1983-06-13 1984-09-11 New England Medical Center Hospitals, Inc. Stabilization of neutrophils and platelets
US4663058A (en) * 1983-10-11 1987-05-05 E. I. Du Pont De Nemours And Company Process for continuous separation of leukocyte/platelet-enriched fraction from whole blood
US4639373A (en) * 1984-11-29 1987-01-27 New England Medical Center Hospitals, Inc. Stabilization of granulocytes
US4923797A (en) * 1988-11-29 1990-05-08 President & Fellows Of Harvard College Stabilization of leukocytes
US5211960A (en) * 1988-11-29 1993-05-18 Scripps Clinic And Research Foundation Stabilization of leukocytes

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US4923797A (en) 1990-05-08
US5369001A (en) 1994-11-29
CA2004043A1 (en) 1990-05-29
WO1990006053A1 (en) 1990-06-14

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