Note: Descriptions are shown in the official language in which they were submitted.
;y;
2015315
Mo-3301-Cont-II-CIP
IMPROVED METHOD OF BLOOD COMPONENT
DECONTAMINATION BY GLUCOSE ADDITION AND
POST-DECONTAMINATION GASSING
BACKGROUND OF THE INVENTION
1. Field of the Invention
Recipients of blood and blood components risk acquiring
infections from foreign biological organisms, either pre-ex-
isting in the blood at the time of collection or transmitted
to the blood product during manipulation. Medical perso~el
who are in contact with collected human blood or clinical
samples also have a significant chance of being exposed to
potentially lethal blood-borne or sample-borne biological
organisms. Blood components today are obtained from blood
donors and frequently involve pooled lots, where one or more
of the donors may be harboring a viral, bacterial or other
infection. Since the blood or blood components are required
to provide physiological functions in a mammalian host, nor-
mally a human host, these functions must not be impaired by
the decontamination treatment of the biological composition.
In addition, the blood or blood components may not be modified
in such a way as to make them immunogenic which could result
~Q15315
in an adverse immune response. Finally, any treatment should
not lease residues or products detrimental to the health of
the host or such residues or products should be readily remov-
able.
2. Description of the Prior Art
U.S. Patent No. 4,327,086 describes the method for heat
treating an aqueous solution containing human blood coagula-
tion factor XIII. U.S. Patent No. 4,321,919 proposes extra-
corporeal treatment of human blood with 8-methoxypsoralen (8-
MOP). Hyde and Hearst, Biochemistry (1978) 17:1251-1257,
describe the binding of two psoralen derivatives to DNA and
chromatin. Musajo et al., Experientia (1965) XXI, 22-24,
describe photo-inactivation of DNA-containing viruses with
photosensitizing furocoumarins. D.S. Patent Nos. 4,350,594,
4,348,283 and 4,350,156 describe filtration methods for selec-
tive removal of blood components based on molecular weight.
U.S. Patent No. 4,329,986 describes extracorporeal treatment
of blood with a chemotherapeutic agent which is subsequently
removed by dialysis. The July/August 1982 issue of Genetic
Engineering News proposed the use of psoralens to sterilize
"clinical or commercial reagents or instruments."
Some data showing substantial impairment of the biological
function of certain enzyme proteins using furocournarins are
published in the scientific literature (see for example, Vero-
nese, F.M. et al., Photochem. Photobiol. 34:351 (1981); Vero-
nese, F.M. et al., Photochem. Photobiol. 36:25 (1982).
~:! ~~ 13315
SUMMARY OF THE INVENTION
Compositions for use in mammalian hosts may be decontami-
nated by treatmen-by furocoumarins and long wavelength ultra-
violet (UVA) light. In this improved process, immediately
following the decontamination, particularly of blood plate-
lets, glucose is added and a post-decontamination gassing may
also be employed in order to minimize risk of damage to blood
platelets.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, compositions to
be employed with mammalian hosts, which may harbor polynucleo-
tides capable of detrimental physiological effects in a host,
are combined with furocoumarin compositians and treated with
WA light under predetermined conditions, whereby the physio-
logical activities of the non-nucleic acid components are
retained. (Whenever the term "polynucleotide" is used in this
application it should be understood to mean: (1) microorgan-
isms containing nucleic acids (either DNA or RNA), (2) nucleic
acid genomes or sub-genomic fragments from microorganisms,
from procaryotes or from eucaryotes, or (3) any other nucleic
acid fragments.)
In decontaminating the biological composition, an aqueous
medium containing the biological preparation is combined with
an appropriate amount of the furocoumarin composition and
-3-
~01~315~
irradiated with ultraviolet light under conditions where all
of the polyr..ucleotide is inactivated, while the components
other than nucleic acid retain their normal physiological
activities.
various biological compositions may be employed, particu-
larly protein compositions involving blood or blood compo-
nents. Whole blood, packed red cells, platelets, and plasma
(fresh or fresh frozen plasma) are of interest. Other blood
components of interest include plasma protein portion, antihe-
mophilic factor (AHF, Factor VIII); Factor IX and Factor IX
complex (Factors IT, VII, IX and X); fibrinogens, Factor XIII,
prothrombin and thrombin (Factor II and IIa); immunoglobulins
(e. g., IgA, IgD, IgE, IgG and IgM and fragments thereof e.g.
Fab, F(ab')2, Fc); hyper-immune globulins as used against
tetanus and hepatitis B; cryoprecipitate; albumin; inter-
ferons; lymphokines; transfer factors; etc. Other biological
compositions include vaccines, recombinant DNA produced pro-
teins, oligopeptide ligands, etc. The protein concentration
in the aqueous medium will generally range from about 1 ug/ml
to 1 gm/ml, more usually from about 1 mg/ml to 100 mg/ml. The
pH will normally be close to physiologic pH
( 7.4), generally in the range of about 6 to 9, more usually
about 7. Other components may be present in the medium, such
as salts, additives, buffers, stabilizers, or the like. These
components will be conventional components, which will be
added for specific functions.
-4-
X015315
The furocoumarins will include psoralen and derivatives,
where the substituent~ will be: alkyl, particularly of i.rom 1
to 3 carbon aton;s, e.g. methyl; alkoxy, particularly of from 1
to 3 carbon atoms, e.g. methoxy; and substituted alkyl, of 1
to 6, more usually 1 to 3 carbon atoms having from 1 to 2
heteroatoms, which will be oxy, particularly hydroxy or alkoxy
of from 1 to 3 carbon atoms, e.g. hydroxymethyl and methoxy-
methyl, or_amino, including mono- and dialkyl amino having a
total of from 1 to 6 carbon atoms, e.g. aminomethyl. There
will be from 1 to 5, usually 2 to 4 substituents, which will
normally be at the 4, 5, 8, 4' and 5' positions, particularly
at the 4'-position. Illustrative compounds include 5-methoxy-
psoralen, 8-methoxypsoralen (8-riOP), 4, 5',8-trimethylpsoralen
(TMP), 4'-hydroxymethyl-4,5'8-trimethylpsoralen (HMT), 4'-
aminomethyl-4,5',8-trimethylpsoralen (FAST), 4-methyipsaralen,
4,4'-dimethylpsoralen, 4,5'-dimethylpsoralen, 4',8-dimethyl-
psoralen, and 4'-methoxymethyl-4,5',8-trimethylpsoralen.
The subject furocoumarins are active with a wide variety
of viruses and other polynucleotides, DNA or RNA, whether
single stranded or double stranded. Illustrative viruses
include: adenovirus, arenavirus, bacteriophage, bunyavirus,
herpesvirus, orthomyxovirus, papovavirus, paramyxovirus, pi-
cornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and
togavirus. Additional pathogenic microorganisms include bac-
teria, chlamydia, mycoplasma, protozoa, rickettsia and other
-5-
i~~ ~?a~~
unicellular microorganisms. Furocoumarins are also effective
in inactivating Hepatitis ~, Non-A Non B Hepatitis, anc AIDS
viruses. This inactivation method may also be uses acainst
uncharacteri2ed infectious agents which cr~av contsin nuc~eic
acid.
In addition to the furocoumarins, additives may be includ-
ed which scavenge for singlet oxygen or other highly reactive
oxygen containing species. Such additives include ascorbate,
glutathione, sodium thionite, etc. In some instances these
additives may have adverse efects, so that in each instance,
their use will be determined empirically. Where such addi-
tives are present, they will be present in amounts ranging
from about 20 ug to 20 mg per ml.
While the process of the earlier applications, and in some
instances patents, works satisfactorily it has been found that
it can be improved. Particularly when blood platelets are
deprived of oxygen, there is some risk of impairment of
cellular respiration. Put another way, there is a risk of
deprivation of the mitochondria of oxygen, causing decreased
aerabic metabolism and a relative increase in glycalysis
resulting in a build-up of lactic acid and a corresponding
decrease of pH. As this pH drop occurs the platelets
deteriorate.
It has been found that two additional steps in the decon-
tamination may be employed, either in combination or individu-
- 6 -
ally in order to minimize the risfi of platelet deterioration.
In particular, if one immediately follows the WA irradiation
with the addition of glucose, the damage to the blood compo-
nents, and particularly to platelets is minimized. The range
of glucose addition should be from about 100 ug/ml to about
100 mg/ml, preferably from about 300 ug/ml to about 1 mg/ml.
When this occurs the evidence, as measured by pH data, shows a
lack of any significant lactic acid buildup. The time of
glucose addition can be immediately following the treatment up
to about 5 hours after the treatment, preferably within the
first hour after treatment.
As an additional improvement step, platelet damage is ,
further minimized if there is a post-decontamination gassing,
preferably with room air. The gassing may also occur with a
combination of oxygen mixed with nitrogen, argon or other
inert gases. One satisfactory method involves flushing with
2 psi air for up to an hour immediately following treatment.
The furocoumarins may be used individually or in combina-
tion. Each of the furocoumarins may be present in amounts
ranging from about 0.01 ug/mI to 1 mg/ml, preferably from
about 0.5 ug/ml to 100 ug/ml, there not being less than about
1 ug/ml nor more than about 1 mg/ml of furocoumarins. Various
psoralen derivatives may be used, including but not limited to
TMP, AMT, HMT, and 8-MOP. 8-MOP and TMP currently are FDA
approved drugs for PUVA therapy in, for example, psoriasis.
Far this reason these compounds are preferred.
_7_
215315
In carrying out the invention, the furocoumarins may be
added to the biological composition by any convenient means in
a manner substantially assuring the uniforr, distribution of
the furocoumarins in the comp~~itior.. The composition ma5~
then be irradiated using WA (320 nm to 400 nm', under condi-
tions ensuring that the entire composition is exposed to suf-
ficient irradiation, so that the furocoumarins may react with
any polynucleotide present to inactivate the polynucleotide.
Depending upon the nature of the medium, particularly its
opacity, as in the case of blood, the depth of the solution
subject to irradiation will vary widely. Usually, the depth
will be not less than about 0.025 millimeter, but may be as
much as a centimeter or more. With whole Dlood, the depth
will generally range from about 0.025 millimeter to 2.5 mil-
limeters. .The light which i~. employed wil3 generally have a
wavelength in the range of about 300 nm to about 400 nm. The
intensity will generally range from about C.1 mW/cm2 to about
W/cm2. In order to prevent denaturation, the temperature
should be maintained below about 60~C., preferably below about
40~~., usually from about -10~C to 30'C. The medium being
irradiated may be irradiated while still, stirred or
circulated, and may either be continuously irradiated or be
subject to alternating periods of irradiation and non-irradia-
tion. The circulation may be in a closed loop system or it
may be in a single pass system ensuring that all of the sample
_g_
~t~)1S:31S
has beer, exposed to irradiation. The total time for irradia-
tior. will vary depending upon the nature of the sample, the
furocoumarin derivative used, the intensity and spectral out-
put of the light source and the nature of the polynucleotides
which may be present. Usually, the time will be at least 1
min. and not more than about 24 hrs., more usually from about
15 mins. to about 6 hrs. When circulating the solution, the
rate of flow will generally be in the range of about 0.1
ml/min to 50 liters/min. It may be desirable to remove the
unexpended psoralen and/or its photobreakdown products from
the irradiation mixture. This can be readily accomplished by
centrifugation, dialysis across an appropriately sized mem-
brane, or ultrafiltration through an appropriately sized hol-
low fiber system. It may be desirable in certain applications
to remove bound or unbound furocoumarins using antibodies,
including monoclonal antibodies, either in solution or at-
tached to a substrate.
The following examples are offered by way of illustration
and not by way of limitation.
_ EgPERIMENTAh
The following experiments were performed in order to dem-
onstrate the ability of the psoralen photoreaction to destroy
microbial contaminants contained in whole blood and blood
products.
g _
2~)1531S
(1~ Feline rhinotracheitis virus, a member of the herpes-
virus ~amily, was added to heparinized whole rabbit blood in
an amount that would give a final concentration of appraxi-
mately 2 X lO~PFU/ml. 4'-hydroxymethyl-4,5',8-trimethylpsora-
len (Hr'T) was added to a portion of the rabbit blood and ali-
quots were irradiated for various periods of time. To test
for remaining live virus, duplicate plaque assays were per-
formed using cultured feline cells (Fc3Tg) (ATCC CCL 176),
with a methylcellulose overlay. Virus titers were obtained as
the arithmetical mean of viral plaques observed in duplicate
assay cultures 72 hours after exposure to test samples. The
results are as follows:
The blood aliquot that received HMT only and no irradia-
tion gave.a titer of 5.3 X 106PFU/ml. The aliguot that re-
ceived HMT and five minutes of irradiation exhibited a titer
of 4.5 X 106 PFU/ml. In the aliquot that received psoralen
plus one hour of irradiation there was no detectable live
virus remaining. The sensitivity of this assay should have
permitted detection of residual virus at titers 1.0 X lOIPFUi
ml. A blood sample which had received HMT and one hour of
irradiation also showed no apparent damage to the red blood
cells as judged by phase contrast microscope analysis and by
absence of visible hemolysis. These data therefore demon-
strate that high virus titers present in whole blood can be
inactivated by psoralen plus light treatment which leaves the
red cell component of the blood intact.
- 10 -
2f115315
(2) In the second experiment Blue Tongue Virus (Serotype
11), a member of the reovirus family, and Feline Rhinotra-
cheitis Virus, and Simian Virus 40 were added to a solution of
Profilate (a commercial preparation of human clotting factor
VIII produced by Alpha Therapeutics). The lyophilized prepa-
ration of Profilate (180 units) was dissolved in 10 ml of
sterile water included with the commercial preparation. This
solution was further diluted with barbital buffer (11.75 g
sodium barbital and 14.67 g NaCl dissolved in 2 liters of de-
ionized water and filtered through a 0.22 micron filter) to a
final concentration of 5 units per milliliter. One portion (2
ml) was set aside at room temperature in the dark. This was
sample #1. A second 2 ml portion was pumped through the
apparatus described below for 1 hour with irradiation. This
was sample #2. Through addition of appropriate amounts of
reagents a'third 2 ml portion was adjusted to contain 10 ug/ml
AMT and 10 ug/ml IiMT and was also irradiated for 1 hour. This
was sample ~3. All the samples were kept at 20~C throughout
the manipulations. The total elapsed time from dissolving of
the lyophilized preparation to the completion of the clotting
factor VIII assays was 6 and one-half hours.
The clotting factor VIII assays were performed at a vari-
ety of dilutions {ranging from 1:5 to 1:100) for each sample
and were compared with the activity in normal human serum and
with pooled normal human serum. The results are summarized in
Table 1.
- 11 -
i~ ~.~i~
TABLE 1
Effect c'_ Photochemical Inactivation
Procedure and Its Components"' on in vitro
Activity of Factor VIII;
Sample
#1 #2 #3
of F- UVA- F- UVA+ F+ UVA+
dilution normal
108 225 150 186
1:5 97 245 155 186
1:10 102 102
1:20 93 92 265 190 232
1:50 101 ~5 255 196 263
1:100 -- 100
254 173 213
Average 98 99
* F = Furacoumarin;
UVA = long wavelength ultraviolet light;
+ Factor VIII activity expressed in $ of normal activity.
100$ = lU/ml of Factor VIII activity
The sample that was subjected to the psoralen inactiva-
tion protocol (sample #3) retained 84$ of the factor VIII
activity that was present in the control sample (#1). This
was higher~than the product activity retained by the sample
that was only irradiated (68~ retained for sample #2) and
indicates that the psoralen photochemistry has little or no
effect on the activity of factor VIII.
Samples otherwise identical to samples 1, 2, and 3 above
were seeded with 2 X 106PFU~m1 of Feline Rhinotracheitis Virus
(FeRT), 1 X 107PFU/ml of Blue Tongue Virus (BTV), and 4 X
208PFU/ml of Simian Virus 40 (SV-40). Table 2 shows the re-
suits of the plaque assays on those samples.
- 12 -
2015315
TABLE 2
Effect of Photochemical Inactivation
Procedure and its Components~ on Infectivity of
Virus in Factor VIII prep2.ration.+
p Sam le 2 Sample 3
Fam ~~1 F- pWA+ F+ UVA+
FeRT Titer 8.6 X 10~ 3.5 X 10~ 0.0
BTV Titer 3.8 X 108 1.9 X 108 1.1 X 103
SV-40 Titer 2.5 X 10 1.6 X 10 1.2 X 10
* F = Furocownarin;
UVA = long wavelength ultraviolet light.
+ Infectivity determined by plaque assays in tissue culture.
In the case of FeRT the number of detectable virus parti-,
cles was reduced by more than five orders of magnitude to
beneath the limit of detection in the plaque assay. The ETV
infectivity was reduced by about five orders of magnitude to
110 PFU/ml. The SV40 infectivity was reduced to a titer of
1.2 X 103. Thus, it is shown that multiple, widely distinct
types of virus can be simultaneously inactivated by at least
five orders of magnitude in the presence of factor VIII, using
the simple, convenient, brief process described above, with
retention of at least 84% of factor VIII activity. Based on
the above observations, it is predictable that by extending,
repeating or modifying the treatment, the probability of an
infectious virus particle remaining can be reduced to an arbi-
trarily Iow value. In this manner suitable safety margins can
be achieved for any of the cited applications.
- 13 -
'~(n.3,a,~ i
APPARATUS AND SYSTEM.
Since whole blood exhibits very high optical density for
longwave UV light (absorption is high for visible light in the
40C nm to 50C nlr. range), the blood was irradiated through a
suitably short optical path length. In this experiment blood
was pumped through polyethylene capillary tubing of 0.875 mil-
limeter inside diameter. The tubing was coiled around a 1.27
centimeter diameter tube and immersed in water which was main-
tained at 18~C. The blood was continuously circulated through
the tubing by means of a peristaltic pump. The blood required
aproximately 2.5 minutes for a complete cycle through the
capillary tubing and was in the light beam for approximately
20$ of the stated irradiation time. The light source was a
low pressure mercury lamp filtered through a cobalt glass
filter. The filter transmits light of approximately 320 nm-
380 nm, with peak transmittance az 360 nm. The incident
intensity at the sample was approximately 40 mW/cm2.
It is evident from the above results, and in accordance
with the subject invention, that polynucleotides in biochemi-
cal compositions can be inactivated to provide a safe composi-
tion for administration to a mammalian host. The proteins
present in the composition retain their physiological activi-
ty, so that they can fulfill their physiological function in a
mammalian host. The method is simple, rapid, and can be ex-
panded to treat large samples. The small amount of chemical
reagent required will not generally be harmful to the host.
- 14 -
~:~1:i315
EXAMPLES OF IP:PROVED ~'lETHOD
OF PLATELET DECONTAI~!INATION
A series of experiments was performed to deterrr,ine inac-
tivation conditions whici: would minimize damage to platelets.
In these experiments, platelet concentrates were treated under
various conditions and stored at room temperature for 96 hrs
following treatment. The platelet integrity was assessed by
pH measurement and morphology evaluation at 24 hr intervals.
As shown in Table 3, UVA irradiation for 10 hrs can be
deleterious to platelets. The pH of the treated platelets
fell from 7.31 to 6.83 immediately following treatment while
the pH of the control remained essentially unchanged. Pro-
longed storage (72 hrs) of treated platelets resulted in a
drop of pH to 5.69. A platelet pH within.the range of 6.7 to
7.4 is consistent with healthy platelets withaut a significant
lactic acid build up.
In an attempt to reduce platelet injury during UVA irra-
diation, any pcssible short wavelength Uv was blocked by irra-
diating through 1/4 inch-thick acrylic sheets. Apparently,
the platelet damage was not due to short wavelength UV. Nith
irradiation -in the presence of acrylic filters, the pH of the
treated platelets dropped just as much (7.31 to 5.67) as with-
out filters within 72 hrs following treatment.
Furthermore, flushing with N2 did not protect platelets
from injury due to 10-hr UVA irradiation, as judged by the pH
- 15 -
215315
decrease (7.28 to 5.73). Accordingly, as shown in Table 4,
the morphology score of 10 hr-WA treated platelets dropped
drastically from 360 to 98-103 ?2 hrs following treatment,
while the control platelets had scores above 200 after 96 hrs
of storage.
The platelet damage due to WA irradiation alone is pro-
portional to the UVA irradiation time. By shortening the
irradiation time to 6 hrs, the pH of the treated platelets
remained essentially unchanged (?.2 to ?.50) immediately fol-
lowing treatment. Long term storage resulted in a decrease
from ?.52 to 6.26. Irradiation under N2 appeared to be advan-
tageous for the shorter time of WA irradiation. As shown in
Table 3, the final pH after 96 hrs of storage is 6.96 if
platelet concentrates were irradiated for 6 hrs under N2.
Accordingly, the morphology scores (Table 4) for the 6 hr-WA
treated platelets were above 200 even after 96 hrs of storage.
These scores are comparable to those of the untreated control
platelets.
Addition of 8-LOOP to the irradiation system had essen-
tially no effect on the pH of the platelet concentrates. As
shown in Table 3, 6 hrs of irradiation under N2 with 300 ug/ml
of 8-MOP resulted in the pH drop from ?.55 to 6.89 after 96
hrs of storage. This is comparable to the pH change obtained
in the absence of 8-MOP (?.54 to 6.96). Furthermore, WA
irradiation in the presence of 8-MOP has minimal additional
- 16 -
zo zsass
effect on the morphology scores of treated platelets (Table
4). Thus it was concluded that UVA irradiation. does have an
effect on platelet concentrates. The effect could be mini-
mized by irradiating in the substantial absence of oxygen.
In this improved process a~aitional measures are taken to
provide further protection for treated platelets from damage
occurring during WA irradiation and subsequent storage.
Glucose is added in these examples to a final concentration of
1 mg/ml to treated platelet concentrates at the end of the
treatment. As shown in Table 3, this appeared to have an
effect on maintaining the pH of platelet concentrates above
7.0 upon prolonged storage. The final pH after 96 hrs of
storage following treatment was 7.11. ,In yet another improve-
ment, since flushing with N2 for 6.5 hrs resulted in an in-
crease of 0.22 to 0.51 units ir. pH of treated platelet concen-
trates, the pH of the treated platelet concentrates can be
brought back to its initial value by exchanging the N2 with
air. This was accomplished by flushing with 2 psi of air for
an hour immediately following treatment. As shown in Table 3,
the final pH of treated platelets after 96 hrs of storage was
7.15. Since both the glucose addition and air exchange had a
positive effect on maintaining the pH of treated platelets
upon storage without lowering their morphology scores (Table
4), these measures can be adopted as routine procedures for an
improved photochemical decontamination protocol.
- 17 -
215315
Finally, in order to eliminate the pH increase due to r~
flushing during Ll~'A irradiation, flushing with 2 psi of 5~e CG2
mixed with N2 was trie~. As showy. in Table 3, the pH of
treated platelet concentrates was 7.38 immediately following
treatment and was 7.40 29 hrs thereafter. Furthermore, the pH
was maintained at 7.30 even after 96 hrs of storage. The
morphology scores {Table 4) of treated platelets were compa-
rable to those of untreated control platelets. Therefore,
subsequent experiments were carried out by flushing with 2 psi
of 5% CO~ and 95% N2.
WA-irradiation under 5% C02 and 95% N2 appeared to allow
longer irradiation time with maintenance of an acceptable
solution pH. Data presented in Tables 3 and 4 shows that
after 8 to 10 hrs of irradiation under 5%'C02, the pH of
treated platelet concentrates was maintained above 7.0 ever.
after Q8 hrs of storage, and the morphology scores were compa-
rable to those of the control sample even at T72.
- 18 -
TABLE 3
Summary of Extracellular pH of Platelet
ConcentratesTreated Under Various Conditions
pH
8-MOP glucose air
TO TE T29 T48 T72 T96
Control - - - 7.54 7.56 7.59 7.61 7.65 7.69
l0hr-WA - - - 7.31 6.83 6.21 5.72 5.69 N.D.
lOhr-WA+acrylic - - - 7.31 6.80 6.24 5.75 5.67 N.D.
lOhr-WA+N2 - - - 7.28 7.50 6.55 5.85 5.73 N.D.
6hr-WA - - - 7.52 7.50 7.28 6.89 6.59 6.26
~~.-WA+N2 - - - 7 .54 ? . 93 7 .41 7 .26 7 .12 6 . 96
6hr-WA+N2 + - - 7.55 7.94 7.38 ?.27 7.10 6.89
6hr-tNA+N2 + + - 7.52 8.03 7.43 7.26 7.20 7.11
6hr-UVA+N2 + + + 7.52 8.01 7.46 7.32 7.23 7.15
6hr-WA+C02+N2 + + + 7.52 7.38 7.40 7.43 7.44 .7.30
8hr-WA+CO +N + + + 7.48 7.26 7.35 7.16 N.D. 6.65
lOhr-WA+Ca2+~2 + + + 7.56 7.27 7.34 7.15 6.95 6.61
8-MOP was used at a final concentration of 300 pg/ml. Glucose was
added at the completion of the treatment to a final concentration of
1 mg/ml. All gas flushing was done at 2 psi. TO = beginning of the
treatment time. Time of treatment for different experiments is speci-
fied in the first column. TE = immediately after the end of treat-
ment. T24, T48, T72 and T96 = 24hrs, 48hrs, 72hrs, and 96hrs after
the beginning of the treatment. WA = irradiation under WA lamps
(peak spectral output from 340 to 380 nm) at an intensity of 3.5 -
4:B mW/cm N - 100$ N2 compressed gas (Liquid Carbonics, San
Carlos, CA). ~0 + N2 = 5$ C02: 95$ N custom-mixed compressed gas
(Liquid Carbonic , San Carlos, CA). a~rylic = 1/4 inch thick sheet of
polyacrylate plastic (TAP Plastics, San Leandro, CA). air = flushing
at 2 psi with compressed air. N.D. = not determined.
- 19 -
TABLE 4
Summary of Morphology Scores of
Platelet Concentrates Treated Under Various Condition
Morphology
Scores
8-MOPglucose
air TE T24 T48 T72 T96
Tp
- - 308 265 297 217 210 218
Control -
- N.D. N.D. 189 130 103 ND
lOhr-WA - - N.D. N.D. 217 145 98 N.D.
-
l0hr-WA+acrylic- - 360 N.D. 217 94 103 N.D.
-
lOhr-WA+N2 - -
- 347 294 269 N.D. 271 211
6hr-WA - - 320 266 255 23? 230 214
6hr-WA+N2 - - _ 309 245 267 236 234 234
6hr-WA+N2 + - - 347 275 276 N.D. 290 '
234
6hr-WA+N2 + + 347 276 280 N.D. 250 232
+
6hr-WA+N2 + +
+ + 264 N.D. 226 273 218 212
6hr-WA+C02+N2 + + 264 N.D. 200 228 N.D. 190
8hr-WA+CO2+N + + 276 N.D. 242 220 219 202
~ + + +
12
l0hr-WA+Co2+
g-MOp was used at a final concentration of 300 ~xg/ml. Glucose was
added at the completion of the treatment to a final concentration of 1
mg/ml. All gas flushing was done at 2 psi. TO = beginning of the
treatment time. Time of treatment for different experiments is speci-
fied in the first column. TE = immediately after the end of treat-
ment. T24, T48, T72 and T96 = 24hrs, 48hrs, 72hrs and 96hrs after the
beginning of the treatment. WA = irradiation under WA lamps (peak
spectral output from 340 to 380 nm) et an intensity of 3.5 - 4.8
mW/cm . N = 100% N compressed gas (Liquid Carbonics, San Carlos,
CA). CO ~ N = 5% ~02 : 95% N2 custom-mixed compressed gas (Liquid
Carbonic , Sa~ Carlos, CA). acrylic - 1/4 inch thick sheet of poly-
acrylate plastic (TAP Plastics, San Leandro, CA). air = flushing at 2
psi with compressed air. N.D. - not determined.
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Although the foregoinc invention has been described in
some detail by way o: illustration and Example fey purposes of
clarity of understandin5, it will be obvious tha:. certain
changes and modifications may be practiced within the scope of
the appended claims.
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