Note: Descriptions are shown in the official language in which they were submitted.
US
Field of the Invention
This invention relates to stabilized control products
useful in the clinical environment and in particular
relates to stabilized coagulation control reagents and
plasma-renin control reagents.
Background of the Invention
This invention is related to a cop ending Canadian apply-
cation Serial No. 455,085 entitled "A Stabilized Multi-
parameter Control Product" (OARED) filed May 25, 1984 by
the inventor hereof.
Hemostasis is a complicated life saving procedure involve
in blood vessels plasma, platelets, vessel walls, neural
and hum oral activity all for the purpose of stanching
bleeding. Although there has been much research,
especially within the last few decades, concerning the
process of coagulation specifically and hemostasis in
general, there is much that is not completely understood
particularly with regard to the specific details concern-
in the interaction of contributing mechanisms. For
instance it is now known that there are no less than 35
compounds which take part in the formation of a firm blood
clot. Ostensibly, blood coagulation is thus one of thy
more complicated chemical processes occurring within the
body In fact, there are so many separate components
within the scheme of coagulation that the International
Committee on nomenclature for Blood Coagulation formed by
the International Hematology Congress has adopted a series
of names which have now come into general acceptance. The
most common of these names (with generally employed
OARED
alternatives) and factor designations include the
following:
Factor I Fibrinogen
Factor II Prothrombin
Factor III Thromboplastin
Factor IV Calcium
Factor V Proaccelerin, labile factor
Factor VI (no longer used)
Factor VII Serum prothrombin conversion
accelerator (SPA), stable factor
Factor VIII Antihemophilic factor (AH)
Factor IX Christmas factor, plasma
thromboplastin component (PTC)
Factor X Stuart Factor, Stuart-Prower
factor
Factor XI Plasma thromboplastin antecedent
(PTA)
Factor XII Hegemony factor
Factor XIII Fibrin stabilizing factor
Profibrinolysin Plasminogen
Fibrinolysin Plasm in
Diagnostic determination of the presence and quantitation
of various of the above factors is important as many
clinically relevant diseases associated with abnormal
clotting are associated therewith. For instance, the
absence of Factor VIII is the most frequent and serious
cause of genetically determined clotting defects such as
hemophilia A which has been recognized for over 2,000
years. A deficiency in plasma concentration of Factor IX
is generally associated with the-genetically determined
defect responsible for hemophilia B. hemorrhagic disease
results from deficiencies in Factors X and XI whereas
deficiencies in Factor XII induce longer clotting times
OR 52
I
but still permit eventual clotting. Diseases of the liver
or Vitamin K deficiencies have also been associated with
abnormal levels of clotting Factors II, VII, IX r and X as
these are predominantly produced by the liver. Other
causes of abnormal clotting may be related to reduced
platelet counts, ~hrombocytopenia, often associated with
abnormal levels of clotting Factors II, VII, IX, and X as
these are predominantly produced by the liver. Other
causes of abnormal clotting may be related to reduced
platelet counts, thrombocytopenia, often associated with
pernicious anemia, certain drug therapies, irradiation or
increased peripheral destruction by antibodies.
The clinical value of testing for coagulation times is not
limited to the detection of genetic or pathological
disease states but is also useful in the regulation of
anticoagulant therapy. Anticoagulants typically inhibit
the coagulation mechanism such as by the heparin mediated
inhibition of Factor X by Antithrombin III. Deficiencies
in the clotting mechanisms due to congenital defects,
pathological conditions, or anti coagulation therapy have
been discussed generally in Chapter 7, "Coagulation and
Hemostasis" by Robert D. Languidly, In Clinical Diagnosis,
Davidson and Henry, 1979.
As commonly understood, coagulation may occur by two path-
ways, the so called intrinsic pathway and extrinsic path-
way. The former is generally triggered by the presence of
a surface (thought to activate Factor XII) and, with the
presence of phospholipids and calcium, through a number of
steps eventually stimulates the formation of a stabilized
fibrin clot. The partial thromboplastin time (PUT) test
typically measures the intrinsic pathway wherein most
congenital deficiencies occur. Consequently this type of
test serves as an excellent presurgical coagulation
screening test; however, since the reagent employed in
OARED
us
this test is typically a platelet substitute, the PUT test
does not measure platelet activity.
The extrinsic pathway is generally triggered by injury to
tissue and the resultant exudation of tissue thromboplas-
tin acts on Factors VII and X which, following a series of
steps, thereafter forms a fibrin clot. The extrinsic
pathway is generally tested by the so called one-stage
prothrombin time test to thereby detect most of the
factors depressed by oral anticoagulant drugs for antic-
agulant therapy control. The thrombin time test typically
measures the quantity and reactivity of fibrinogen. This
test is a rapid, semi quantitative test and is consequently
the test of choice for intravascular clotting and
fibrinolysis analysis. The above is presented in review
in accompanying Table I as well as a booklet entitled
"Concentric Concepts of Coagulation 1971, 1975" available
from Ortho Diagnostics, Inc., Raritan, New Jersey.
OARED
I
--5--
o
N t_)
C:
C.,1 I
I:: 9
Of:; Us S
lo IJ A
O
Z I 1
H O P'
Q 1_1
H
I) O H
as X
I H Eel Us ^ A
P' En I a
Clue
H O E-l r
So O
H E I Jo
H I
X p
î a
C
a .-1
0
Jo .
Q j Eon
I
Jo
Q I
, _
En
î c us
.~,
I:
O
~1
Us I Go
J C
.,, I O
Q. I Jo
Jo I Us
I/
O
Us O
O I J-
S I
+
or En
A
Us X I
us J I I I to
H - H I
Us Jo > H -1 1 I c
I w pa x X Al
I
Al I
Z Do
H E-l Us 5.1
O
.,1 Q _
So O I -
O
E-l E I P ;
With such attention being placed on the determination of a
particular patient's coagulating ability and the presence
or absence of specific factors, it is axiomatic that
adequate controls must be available in order to ensure the
proper operation of manual and automated methods designed
to determine these factor levels and coagulation in
general. Heretofore, such controls as were available have
been typically characterized by relatively short shelf
fifes both in the liquid and dry state due largely, in
part, to the unstable nature of the ingredients or by
their inability to withstand drying procedures such as
lyophilization.
It us an object of the present invention to provide
plasma-renin and coagulation control reagents having the
appropriate levels of particular factors present therein
in a stabilized format and capable of being lyophilized.
It is another object of the present invention to provide
methods whereby coagulation control and plasma-renin
control reagents may be stabilized
It is another related object of the present invention to
provide reagents in a stabilized format useful in manual
and automated renal hypertensive testing and coagulation
testing.
Brief Summary of the Invention
In accordance with the objects and principles of the
present invention, coagulation control reagents and plasma
resin control reagents are provided which are
substantially stabilized by the addition of plexiform
stabilizing means. In the case of coagulation control
reagents, anti coagulation means is added to substantially
defibrinated plasma, whereby clotting components are held
OARED
intact and residual fibrin formation is inhibited.
Additionally, plexiform stabilizing means, selected from
the group consisting of sucrose, reducing monosaccharide
sugars and reducing disaccharide sugars, is added so that
S it is present in the final product in he range of about
3%-10% volume percent The preferred plexiform
stabilizing means is selected from the group consisting of
maltose, minutely, syllabus, glucose and lactose with
the latter most being the most preferred. Additionally,
and in accordance with the typo of coagulation control
reagent desired, a specific clotting factor or factors may
be removed such as Factor VIII and/or Factor I.
The plasma resin control reagent of the present invention
comprises selected plasma to which has been added
anti coagulation means whereby clotting components are held
substantially intact, and plexiform stabilizing means
similar to that described for the coagulation control
reagent. The anti coagulation means is ideally chosen from
eye group consisting of citrate, oxalate, ETA (ethylene
Damon tetraacetic acid), heparin, and any combination of
the foregoing.
The most preferred embodiments will have substantially all
water removed by processes well known such as by
lyophilization~
In another embodiment the present invention provides a
method for producing a stabilized coagulation control reagent
comprising the steps of: (a) providing whole blood; (b)
removing substantially all cells from said whole blood to
obtain plasma; (c) adding anti coagulation means whereby
clotting components are held intact and fibrin formation
is inhibited; (d) adjusting specific clotting Factor or
Factors in accordance with the type of coagulation control
reagent desired, (e) adding plexiform stabilizing means
-pa-
selected from the group consisting of sucrose, reducing
monosaccharide sugars and reducing disaccharide sugars
so that said plexiform stabilizing means is present in the
range of about 3% to 10% final volume percent,
In still another embodiment the present invention provides
a stabilized plasma resin control resent useful for Anglo-
tension testing comprising: (a) plasma having a known resin
level; (b) anti coagulation means whereby clotting components
are held intact and fibrin formation is substantially
inhibited; and (c) plexiform stabilizing means selected
from the group consisting of reducing monosaccharide sugars
and reducing disaccharide sugars whereby said plexiform
stabilizing means is present in the range of about 3% to
10% final volume percent.
The reagent products of the present invention are useful
for use in manual methods as well as automated methods in
chemistry analysis and are particular useful for those
2 tests involving coagulation and plasma resin activity.
These tests include, for instance, renal hypertension
testing, actual coagulation testing, testing for
angiotension I Andre II, and resin activity testing
'7~5i
which is typically run only on fresh human plasma. As may
be expected, particularly with the latter most test,
adequate control reagents are difficult to obtain
particularly in a suitably stabilized format.
s
Such a stabilized format is particularly desirous in the
clinical environment in order to minimize cost
expenditures and to provide increased comparative
integrity of the data on a daily testing basis.
Ideally, the control reagent materials will simulate a
patient's sample as closely as possible except, to the
extent the controls may be artificially altered in order
to provide factor deficient controls to levels which may
be reasonably expected with particular disease states.
For instance, a patient suffering a cardiac infarction may
be expected to have increased levels of resin. Resin is
known to act on plasma proteins thereby forming
angiotensin, in turn, responsible for an increase in blood
pressure occasioned by constricted blood vessels. The
resin, a proteolytic enzyme believed to be produced in the
efferent golmerular arterioles, reacts with
hypertensinogen to product angiotensin II. The latter is
a decapeptide hormone which, in addition to influencing
US blood vessel constriction, also alters aldosterone
secretion by the adrenal cortex. Angiotensin is also
known as hypertension. Aldosterone is a steroid hormone
which function chiefly in regulating sodium and potassium
metabolism and thereby it also plays an important part in
cardiac function.
Accordingly, and in order to ensure accurate testing
procedures, a plasma resin control reagent is a virtual
necessity, and is particularly desirable if made in a
stable format. In accordance with the objects of the
present invention, such a control reagent is now made
OARED
7~5
g
hereby possible. It comprises plasma, ideally from human
origin, preferably selected for its high resin levels.
Additionally, an anticoagulant is added whereby the
clotting components may be held intact and fibrin
formation inhibited. Such an anticoagulant may ye
selected from the group consisting of Satyr oxalate,
ETA, heparin or a combination thereof.
The desired stability is obtained by adding plexiform
stabilizing means selected from the group consisting of
reducing monosaccharide sugars and reducing disaccharide
sugars Preferred embodiments employ maltose, minutely,
syllabus, lactose or glucose. The plexiform stabilizing
means is added so that the final percent concentration is
preferably in the range of about 3%-10%. As more fully
described in the OARED application Serial Noah referenced
earlier, the plexiform stabilizing means is thought to
provide a three dimensional crystalline" structure
whereby the proteins, analyzes, and constituents of the
control reagents are held in stabilized juxtaposition. It
is further believed by the inventor hereof, although he
does not wish such belief to be a limitation hereof, that
the plexiform stabilizing means serves to hold the
individual constituents in a substantially rigid manner
thereby reducing the amount of degradation or lability
which may be associated or occasioned by the physical
movement of the analyzes.
Further, the plexiform stabilizing means serves to assist
in the sublimation of water during water removal processes
such as by lyophilization. The actual mechanism by which
this is accomplished is unknown, however, it has been
observed, particularly with respect to the clinical
OR 52
-10-
chemistry control reagents of the OARED application, that
reconstitution of the reagent after lyophilization is
accomplished with greater speed and with increased vial-
to-vial reproducibility These advantages may also be
expected following lyophilization of the control reagents
of the present invention.
Similarly, the coagulation control reagents are ideally
produced from plasma having added thereto an anticoagulant such
as that described above, whereby the clotting components
are held intact and residual fibrin formation is
substantially inhibited. In addition to supplementation
with the above-mentioned plexiform stabilizing means, the
coagulation control reagent may be adjusted to remove
specific clotting factor or factors in accordance with the
type of coagulation control reagent desired. For
instance, should a reagent be desired which is sub Stan-
tidally deficient in Factor I ~fibrinogen~ or Factor VIII
(antihaemophelic factor), then those components may be
removed such as by ethanol extraction or chilling,
respectively, or by other methods for other Factors as is
well-known in the art.
As with the plasma resin control reagent, the plexiform
stabilizing means acts to provide a three dimensional
structure to greatly increase stability, speed of
reconstitution following lyophilization and superior vial-
to-vial reproducibility. Further, and as is more fully
explained in the OARED application SUN Thea speed with which
lyophilization is accomplished may actually be increased
despite the lowering of the freezing point This is
thought to be generally due to the of imitation of the
eutectic point plateau. Additionally, and for reasons not
yet fully explained, the reconstituted materials typically
exhibit greater optical clarity
OARED
This is probably due, at least in part, to the decreased
denaturation of proteins, particularly a problem during
lyophilization.
Still other advantages are gained by the instant invention
and include the observation that the defibrinated plasma
appears more completely defibrinated with the addition of
the plexiform stabilizing means than defibrinated plasma
not so augmented. The observed, more consistent gel-like
clot may thus be a natural progression therefrom and may
also be indicative of the enhanced overall stability of
other inherent clotting factors present in the plasma.
The most preferred reagents of the present invention will
also have substantially all water removed by processes
well-known in the art. Such processes include, without
limitation, lyophilization and the like, and serve to
afford even greater shelf lives. Ideally, the resultant
dry product is stored at approximately 2- 8C and
reconstituted just prior to use.
Stability as used herein shall mean later measured
activity which is at least 90~ of the original measured
activity.
As may be readily appreciated by those skilled in the art,
numerous insubstantial alterations or substitutions of the
aforementioned components may be made without departure
from the spirit or scope of the present invention.
OARED
