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Patent 2096212 Summary

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(12) Patent Application: (11) CA 2096212
(54) English Title: INITIATOR OF THE INTRINSIC COAGULATIONOF BLOOD, PLASMA OR BLOOD DERIVATIVES, A REAGENT FOR DETERMINING THE APTT OF BLOOD, PLASMA OR BLOOD DERIVATIVES, A METHOD OF PRODUCING THIS REAGENT AND A METHOD OF DETERMINING THE APTT OF SAMPLES OF BLOOD, PLASMA OR BLOOD DERIVATIVES
(54) French Title: DECLENCHEUR DE LA COAGULATION INTRINSEQUE DU SANG, DU PLASMA OU DE DERIVES SANGUINS, REACTIF POUR LA DETERMINATION DE APTT DANS LE SANG, LE PLASMA OU LES DERIVES SANGUINS, MODE DEPRODUCTION DE CE REACTIF ET METHODE DE DETERMINATION DE APTT DANS DES ECHANTILLONS DE SANG, DE PLASMA OU DE DERIVES SANGUINS
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12Q 1/56 (2006.01)
  • G01N 33/86 (2006.01)
(72) Inventors :
  • LANG, HARTMUT (Austria)
  • MORITZ, BERTA (Austria)
(73) Owners :
  • IMMUNO AKTIENGESELLSCHAFT (Austria)
(71) Applicants :
(74) Agent: FETHERSTONHAUGH & CO.
(74) Associate agent:
(45) Issued:
(22) Filed Date: 1993-05-13
(41) Open to Public Inspection: 1993-11-16
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
A 998/92 Austria 1992-05-15
A 841/93 Austria 1993-04-30

Abstracts

English Abstract



ABSTRACT OF THE DISCLOSURE

An initiator of the intrinsic coagulation of
blood, plasma or blood derivatives is claimed, which is
characterised in that it comprises a mixture of
sulfatides and a solid activator, preferably kaolin.
Furthermore, a reagent for determining the aPTT of
blood or blood derivatives is claimed, which comprises
a mixture of sulfatides and a solid activator,
preferably kaolin, as the initiator of the intrinsic
coagulation, and which furthermore contains
phospholipids.
The invention also relates to a reagent for
assaying the aPTT of blood, plasma or blood derivatives
possibly containing heparin or heparinoid and
containing an initiator of the intrinsic coagulation,
which is characterised in that it contains a heparin-
neutralizing agent and phospholipids. A method of
assying the aPTT in such samples as well as the use of
this method for assaying the activity of factors of the
intrinsic coagulation and the inhibitors thereof is
also claimed.


Claims

Note: Claims are shown in the official language in which they were submitted.


WHAT WE CLAIM IS:


1. An initiator of the intrinsic coagulation of
blood, plasma or blood derivatives comprising a mixture
of sulfatides and a solid activator.

2. An initiator as set forth in claim 1, wherein said
solid activator is kaolin.


3. A reagent for determining the aPTT of blood,
plasma or blood derivatives comprising a mixture of
sulfatides and a solid activator as initiator of the
intrinsic coagulation, and furthermore comprising
phospholipids.

4. A reagent as set forth in claim 3, wherein said
solid activator is kaolin.

5. A reagent as set forth in claim 3, wherein said
phospholipids are chemically pure phospholipids in a
standardized mixture.


6. A reagent as set forth in claim 5, wherein said
phospholipid mixture, based on the total phospholipid
content, comprises 5 to 50 % of phosphatidyl serine, 15
to 60 % of phosphatidyl choline, 15 to 60 % of
phosphatidyl ethanol amine, 0 to 20 % of cholesterol



- 22 -

and 5 to 20 % of phosphatidinic acid.

7. A reagent as set forth in claim 3, further
comprising at least one of oxidation inhibitors and
sensitivity promoters.


8. A reagent as set forth in claim 3, further
comprising alkali salts.

9. A reagent as set forth in claim 8, further
comprising amino acids.

10. A reagent as set forth in claim 3, further
comprising a chromogenic substrate enabling a
photometric determination of the coagulation time by
colour development.


11. A reagent as set forth in claim 3, further
comprising at least one of stabilizers and buffer
substances.

12. A reagent as set forth in claim 11, wherein among
said stabilizers is albumin.

13. A reagent as set forth in claim 3 in the
lyophilized form.


- 23 -

14. A method of producing a reagent for determining
the aPTT of blood, plasma or blood derivatives, said
method comprising
providing sulfatides,
providing an aqueous suspension of a solid activator,
providing phospholipids,
mixing said sulfatides with said phospholipids in said
aqueous suspension of said solid activator.



15. A method as set forth in claim 14, further
comprising
providing further additives, and
mixing said further additives with said sulfatides in
said aqueous suspension of said solid activator.



16. A method as set forth in claim 14, further
comprising
lyophilizing said reagent.



17. A method of producing a reagent for determining
the aPTT of blood, plasma or blood derivatives, said
method comprising
providing sulfatides,
providing a solid activator,
providing phospholipids and ,
mixing said sulfatides, said phospholipids, and said
solid activator in the solid state.



- 24 -

18. A method as set forth in claim 17, further
comprising lyophilizing said sulfatides, said solid
activator and said phospholipids before mixing them.



19. A method as set forth in claim 17, further
comprising providing further additives, and
admixing said further additives.



20. A method of determining the partial thromboplastin
time (aPTT) of samples of blood, plasma or blood
derivatives, said method comprising
providing a sample,
providing a reagent comprised of a mixture of
sulfatides and a solid activator as initiator of the
intrinsic coagulation, and of phospholipids,
contacting said sample with said reagent for a pre-
determined activation time,
admixing calcium ions after said pre-determined
activation time, and
determining the coagulation time of said sample.



21. A method as set forth in claim 20, further
comprising
providing a heparin-neutralizing substance, and
admixing said heparin-neutralizing substance with said
sample before contacting said sample with said reagent.


- 25 -


22. A method as set forth in claim 20 to be used for
determining the activities of factors of the intrinsic
coagulation and inhibitors thereof, wherein a plasma
sample is contacted with said reagent, calcium ions are
admixed after a pre-determined activation time, and the
coagulation time is determined so as to give a certain
result, and said certain result is compared to the
result of a standard sample of known activity of the
factors.

23. In a reagent for determining the aPTT of blood,
plasma or blood derivatives containing an initiator of
the intrinsic coagulation, the improvement wherein said
reagent contains a heparin-neutralizing agent and a
phospholipid component.


24. A reagent as set forth in claim 23, wherein said
blood, plasma or blood derivatives contain heparin.

25. A reagent as set forth in claim 23, wherein said
blood, plasma or blood derivatives contain heparinoid.


26. A reagent as set forth in claim 23, wherein said
heparin-neutralizing agent is selected from the group
consisting of polybrene, a protamin salt or heparinase.


- 26 -


27. A reagent as set forth in claim 23, wherein said
phospholipid component, based on the total phospholipid
content, comprises from 5 to 50 % phosphatidyl serine,
from 15 to 60 % phosphatidyl choline, from 15 to 60 %
phosphatidyl ethanol amine, from 0 to 20 % cholesterol
and from 5 to 20 % phosphatidinic acid.



28. In a method of determining the partial
thromboplastin time (aPTT) of samples of blood, plasma
or blood derivatives, the improvement comprising
providing a sample,
providing a reagent containing an initiator of the
intrinsic coagulation, a heparin-neutralizing agent and
phospholipids,
contacting said sample with said reagent,
providing calcium ions,
admixing said calcium ions, and
determining the coagulation time of said sample.



29. A method as set forth in claim 28, wherein said
sample of blood, plasma or blood derivatives contains
heparin.



30. A method as set forth in claim 28, wherein said
sample of blood, plasma or blood derivatives contains
heparinoid.


- 27 -


31. A method as set forth in claim 28 to be used for
determining the activities of factors of the intrinsic
coagulation and inhibitors thereof, wherein a sample of
blood, plasma or blood derivatives is contacted with
said reagent, calcium ions are admixed after a pre-
determined activation time, and the coagulation time is
determined so as to give a certain result, and said
certain result is compared with the result of a
standard sample of known activity of said factors.

32. A method as set forth in claim 31, wherein said
sample of blood, plasma or blood derivatives contains
heparin.

33. A method as set forth in claim 31, wherein said
sample of blood, plasma or blood derivatives contains
heparinoid.

34. A set comprised of
a) a reagent for assaying the APTT and comprising an
initiator of the intrinsic coagulation and
phospholipids, and
b) a heparin-neutralizing agent.

- 28 -

Description

Note: Descriptions are shown in the official language in which they were submitted.




The invention relates to an initiator of the
intrinsic coagulation, to a reagent for determining the
activated partial thromboplastin time (aPTT) as well as
to a method of producing this reagent and of
determining the aPTT.
The activated partial tromboplastin time o~ a
plasma sample serves as screening test for the overall
determination of the activity of the coagulation .
factors of the intrinsic coagulation. Disorders in the
coagulation course cause coagulation times that lie
beyond the normal range. An increased aPTT, as occurs
e.g. in hemophilia A, manifests a deficiency of
coagulation factor VIII, e.g.. In addition, the aPTT
test i9 frequently used for monitoring a heparin
therapy.
A reagent for determining the aPTT contains an
activator to initiate the cascade of the intrinsic
coagulation up to the formation of activated factor IX.
Furthermore, phospholipids are required for the
activation of factor X and of prothrombin, and there-
fore they are also contained in an aPTT reagent. The
reagent is contacted with a plasma sample, whereupon
calcium is added after a defined activation time which
is to be as short as possible, and the time until
formation of a fibrin clot is measured.
The aPTT ~est system is subject to a plurality of
requirements. For routine check-ups, easy handling of


-- 2



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. ,: , ~ .

c~ 2 ~ ~


an aPTT reagent is an abolute prerequisite. Also, the
stability of the ingredients must be very high to
ensure a constant coagulation time over a long period
of time. This also enables a low-cost utilisation of
the reagent. For the aPTT as a general test, a balanced
sentitivity relative to all the pa:rameters to be
registered is necessary.
Common reagents *or determining the aPTT contain
surface active solid as the initiator of the intrinsic
coagulation. By using a surface-active matrix, at first
factor XII is activated, comparable to the physiologic
conditions. As thA solid activator, e.g. kaolin, celite
and glass are used. Kaolin, e.g., is contained as
suspension in commercially common reagents for
determining the aPTT (Pathromtin~, Behringwerke (1990~,
or PTT Reagent, Boehringer Mannheim (1986)). As the
phospholipid, a lyophilised lipid extract of human
placentas or lyophilised cephalin are used. However,
the phospholipids are mixed with kaolin -to a ready-to-
use reagent jus-t before the determina-tion is carried
out. The separate storage of kaolin and phospholipids
is mainly required for stability reasons.
When lyophilizing lipids with kaolin, lipids would
become inactivated to a large extent. Thus, in the
production of PTT-IMMUN0 (of Immuno) there occurs a
loss of up to 50~ of the phospholipid activity at the
common lyophilization of kaolin and phospholipids

;

'~ 2 ~ ~

(extracted from porcine brain).
As an al-ternative to solid activators, also liquid
activators, such as ellagic acid and its derivatives,
or solubilized sulfatides may be used. The sen~itivity
of a liquid-activator-containing test relative to
heparin is, however, deteriorated, and therefore
diverse additives have been suggested to improve its
sensitivity. According to WO 91/16453, a heparin-
sensitive reagent based on ellagic acid additionally
contains dextran sulfate.
A disadvantage of kaolin and of solid activators
in general and of ellagic acid or the derivatives
thereof is the long activation time of the first phase
of the intrinsic coagulation, by which a relatively
long incubation time of at least 4 min is necessary for
activation. A further disadvantage consists in the
relatively long coagulation time in the presence of
kaolin. However, a higher kaolin concentration in the
reagent can shorten the activation time. Yet kaolin,
which deposits from the suspension, may lead to an
increased coagulation time or lead to false results
when determining the end of coagulation by means of the
usual coagulometers. Thus, care must be taken to keep
the solid activator concentration as low as possible.
The invention has as its object to provide a
standardized rPagent with high stability for
determining the aPTT, which is easy to handle and




,', " " '

: - ' ~ " . '

~? 3~ 9 ~

inexpensive and which involves both short activation
times and a high sensitivity relative to individual
factors of -the coagulation cascade.
According to the invention, the set object is
achieved by an initiator of the intrinsic coagulation
of blood, plasma or blood derivatives, which contains a
mixture of sulfatides and a solid activator, preferably
kaolin.
As initiator of the intrinsic coagulation, the
reagent according to -the invention contains a mixture
of sulfatides (e.g. of Pentapharm) and a solid
activator . Furthermore, phospholipids are contained.
The activation time which is only short when using
sulfatides for the activation of factor XII, is hardly
increased by their being used together with kaolin.
Thus, by using the reagent according to the invention,
a procedure of short incubation time practicable for
routine check-ups is made feasible. Simultaneously,
however, also the heparin sensitivity is improved by
the use of the solid activator.
The use of the initiator according to -the
invention in an aPTT reagent thus surprisingly enables
a combination of the advantages of both individual
activators, yet without a combination of their
drawbacks.
This could not be foreseen. Much rather it was to




.

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be surmised that the combination of the sulfatides with
kaolin as initiator of the intrinsic coagulation would
lead to a reduction of ac-tivity in the reagent.
Following the specialists' prejudice that a combination
of activators having different reaction mechanisms
would create problems, it came as a surprise that the
reagent according to the invention can be standardized
very well. This is a precondition for a high
reproducibility of the determining method.
In the reagent according to the invention the
amount of kaolin is kept small. An increased deposit of
kaolin is prevented, problems in the operation of
de~ection apparatus are avoided.
The initiator of the inven-tion may be lyophilized
together with phospholipids without any problems to
give a low-cost reagent. Any possible negative
interaction of the individual components is without any
relevance.
The invention thus relates to a reagent for
determining the aPTT of blood, plasma or blood
derivatives, which is characterised in that it contains
a mixture of sulfatides and a solid activator,
preferably kaolin, as initiator of ~he intrinsic
coagulation, and further contains phospholipids.
The concentrations of kaolin and sulfatides in the
reagent are between 0.1 and 0.6 % by weight, preferably
0.1 to 0.3 % by weight, for k~olin, and between 0.001


2 ~ ~3J


and 0.03 % by weight, preferably 0.002 to 0.01 % by
weight, for sulfatides.
The reagent preferably contains chemically pure
phospholipids in a standardized mixture. It has been
shown that this results in an unexpectedly high
reproducibility of the test method. The phospholipid
mixture comprises, e.g., phosphatidyl serine,
phosphatidyl choline and cholesterol.
The mixture preferably comprises from 5 to 50
phosphatidyl serine, from 15 to 60 ~ phosphatidyl
choline, from 15 to 60 ~ phosphatidyl ethanol amine,
from 0 to 20 % cholesterol and from 5 to 20 %
phosphatidinic acid, based on the total phospholipid
content. As standardized mixture, e.g. 0.2 mg
phosphatidyl serine, 0.85 mg phosphatidyl choline, 0.65
mg phosphatidyl ethanol amine, 0.15 mg cholesterol and
0.15 mg phosphatidinic acid per ml, possibly in
dilution, are used.
Ad~antageously, the phospholipids are purified in
a manner that undesired precipitation reactions, which
may occur in the presence of polybrene, are avoided.
A preferred embodiment of the reagent is
characterised in that it further contains oxidation
inhibitors and sensitivity promoters. ~s the oxidation
inhibitors, e.g., cystein, uric acid, vitamin C or
vitamin E are utilized so as to increasa the storage
stability of the reagent in the lyophilized or liquid




- 7 -

.


state. Additives, such as alkali salts, possibly
together with amino acids, increasle the sensitivity of
the test relative to individual factors. I-t has been
shown that a reagent which contains cesium chl~ride
(from 5 to 25 mg/ml) and arginin (from 2.5 to 20
mg~ml), preferably 16.8 mg of CsCl/ml and 10.5 mg
arginin/ml, after an appropriate dilution, is
particularly well suited for determininy the aPTT of a
plasma sample with a deficiency of individual factors
(factor VIII).
A further preferred embodiment of the reagent is
characterised in that it further contains a chromogenic
substrate, by which, if desired, the coagulation time
can be determined photometrically by color development.
However, the final product can also be determined by a
photometric determina~ion of the change in light
transmission.
Advantageously, the reagent further contains
stabilizers, preferably albumin, and/or buffer
substances.
The method of producing this reagent is simple.
Sulfatides are mixed in an aqeuous suspension of the
solid activator with phospholipids, and, if desired,
with further additives, and, if desired, are
lyophilised.
The production of the reagent may, however, also
be effected by mixing sulfatides, the so}id activator




-- 8 --




. . . .
.



and phospholipids, if desired after lyophilizing,
possibly with fur~her additives, :in the solid state.
The reagent according to the invention lends
itself both to the monitoring of a heparin therapy and
to determining individual factors" i.e. coagulation
i factors or inhibitors of the intrinsic coagulation.
The invention furthermore re]Lates -to a method of
determining the partial thromboplastin time (aPTT) of
samples of blood, plasma or blood derivatives, which is
characterised in that the sample is contacted with a
reagent of the invention, calcium ions are added after
a pre-determined activation time, and the coagulation
time is determined.
By the addition of heparin-neutralizing
substances, a deficiency of coagula-tion factors can
also be determined in heparinized plasma samples.
Polybrene, e.g., neutralizes heparin present and thus
; makes the reagent of the invention insensitive relative
to a heparin content of the sample.
A further preferred embodiment of the method
according to the invention is also characterised in
that the sample is admixed with a heparin-neutralizing
substance~ such as polybrene, protamin sulfate etc.,

. .. .
before the reagent of the invention is added.
Furthermore, the invention relates to the use of a
method according -to the invention for determinin~ the
activity of factors of the intrinsic coagulation and




_ g _




,
'




the inhibitors thereof, wherein a plasma sample is
contacted with a reagent according to the invention,
calcium ions are added after a pre-determined
activation time, and the coagulation time is
determined, whereupon the result obtained is compared
to a standard sample with a known activity of the
factors.
Furthermore, it was found that assaying of t~e
aPTT of blood, plasma or blood derivatives possibly
containing heparin or heparinoid, can also be carried
out without using a mixture of sulfatides and solid
activator, if a heparin-neutralizing agent and
phospholipids are contained in a reagent for assaying
the aPTT, in addition to an initiator of the intrinsic
coagulation, be it a solid activator or a liquid
activator or a combination thereof.
As the heparin-neutralizing agents, in particular
polybrene, protamin (protamin salts) or heparinase are
used in the reagent according to the invention.
As regards the order of magnitude, the
concentrations of the heparin-neutralizing agents used
depend on the heparin concentration in the sample to be
assayed, and usually it ranges between 1 and 500 mg/ml
in the case of polybrene. In the standardized method,
between 2 and 100 mg/ml polybrene are used.
The advantage of a reagent having a heparin-
neutralizing agent consists in particular in that the




-- 10 --

3 ~ ~ 2

assaying of the aPTT and of -the individual factors in a
blood or plasma sample may be effected irrespective of
whether or not the sample contains heparin or
heparinoids. ,
The invention further r~lates to a set comprised
of a) a reagent for assaying the aPTT and comprising an
initiator of the intrinsic coagulation and
phospholipids, and b) a heparin-neutralizing agent.
Although the aPTT is often utilized for monitoring
a heparin therapy, wherein a heparin sensitivity is
desired, there are also cases in which the intact
intrinsic system or factors of the intrinsic system
must be assayed independently of the heparin content.
If heparin were contained in the same and thus the
aPTT were delayed when measured, too low a portion of
coagulation factors would be simulated.
The invention will be explained in more detail by
the following examples.
1. Comparison of the aPTT when using diverse
reagents
As initiator, the assayed reagents for determining
the aPTT contained sulfatides (of Pentapharm) and
kaolin in various mixing ratios in 200 mM Hepes-glycin-
buffer ~pH 6.8). Furthermore, a mixture comprising 4.8
~g phospha~idyl serine, 20.7 ~g phosphatidyl choline,
15.9 ~g phosphatidyl ethanol amine, 3.7 ~g cholesterol
and 3.7 ~g phosphatidinic acid per ml was present in


i 2 ~ ~

the reagent.
0.1 ml normal plasma (NP) (Reference Plasma 100 %,
Immuno) or abnormal plasma (AP) (Immunocontrol Plasma
Abnormal, Immuno) were admixed with 0.1 ml re~gent.
After an incubation time of 2 minutes at 37C, 0.1 ml
0.025 M CaCl2 solution was added, and the time until
the point of coaguation was measured by a Schnitger-
Gross-coagulometer (of Amelung).
From Table 1 it can be seen that the presence of
sulfatides in the reagent shortens the coagulation time
of normal pl.asma (NP). Yet kaolin in the reagent also
improves the sensitivity, expressed by the ra-tio of the
coagulation times of AP and NP (cf. Table 2).
Table 1: aPTT (s)
kaolin (%) 0 0.15 0.30
sulfatide (~)
NP 0 X 78.1 70.1
NP 0.005 34.4 38.4 38.6
NP 0.01 39.1 41.1 41.8



Table 2: Ratio of the aPTT
(AP/NP)
kaolin (%) 0 0.15 0.30
sulfa-tide (~)
0 X 2.20 2.12
0.005 2.00 2.11 2.21
0.01 2.01 2.15 2.~3




- 12 -


2. Sensitivity to heparin
By using the reagents from 1., normal plasma (cf.
1.) and heparinized normal plasma (0.37 U of
heparin/ml) were assayed. In Table, 2, the rat'io of the
aPTT of heparinized normal plasma to normal plasma is
indicated for the different reagents. The sensitivity
relative to heparin is improved by a higher kaolin
content in the reagent.



Table 3: Ratio of the aPTT
~ heparin)
kaolin (~) 0 0.15 0~30
sulfatides (~)
0 X 1.50 1.66
0.005 1.18 1.28 1.37
0.01 1.23 1.31 1.41
3. Sensitivity relative to factor VIII
:
By using the reagents from 1., normal plasma (cf.
1.) and Control Plasma (of Immuno, contained < 30~ of
factor VIII, based on the factor VIII content of normal
plasma (= 100 ~)) were assayed. In Table 4, the ratio
of the aPTT of the Control Plasma to normal plasma is
indicated for the different xeagents. The sensitivity
relative to factor VIII is improved by the content of
sulfatides in the kaolin-containing raagent.




- 13 -
'

. ,:
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Table 4: Ratio of the aPTT
Control Plasma (< 30 % of factor VIII/normal plasma)
kaolin (~) 0 0.15 0.30
sulfatides (%) ',
0 X 1.48 1.51
0.005 1.66 1.75 1.74
0.01 1.71 1.72 1.70
4. Factor VIII-calibration curve
To draw a calibration curve for factor VIII,
Reference Plasma 100 ~ (of Immuno) was diluted 1 : 5
with imidazole buffer 3.4 g/l imidazole, 5.85 g/l NaCl,
pH 7.4). 0.1 ml o~ this dilution were mixed with 0.1 ml
of a phospholipid-kaolin-sulfatide reagent (aomposition
of the phospolipids as in Example 1; 0.3 ~ kaolin and
0.01 % sulfatides in 200 mM Hepes-glycin-buffer, pH
6.8) and incubated for 4 min at 37C. Simultaneously
with the addition of 0.1 ml of calcium chloride (25mM),
the stop watch was started, and the time until thè end
of coagulation was determined by means of a Schnitger-
Gross-coagulometer (of Amelung).
The prediluted plasma (1:5) corresponds to lO0 %
of factor VIII. Further dilutions were tested according
to a geometric dilution series ( 1 : l = 100 ~ to 1 :
128 = 0.78 %). In Table 5, the results of the
measurements are indicated.




.~ 1'1


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Table 5:
- Factor VIII (%) Coagulation Time (s)
lOO 78.5
: 50 91.~ ,
:L01.7
12.5 :Ll5.3
6.25 :L28.9
3.13 .L39.g
:~ 1.56 148.6
:~ 0.7~ 159.4
5. aPTT assaying with PTT reagents of different
compositions, with and without the addition of
~; polybrene.
Five different reagents are used in the examples
: or assaying the aPTT, both with and without the
J addition of heparin-neutralizing agents.
These reagents have the following compositions (in
by weight):
Reagent A: 0.075 % kaolin and 0.01 % sulfatide (of
:~ Pentapharm) as the initiator, highly purified
phospholipids (17 ~ phosphatidyl serine, 44 %
phosphatidyl choline, 29 % phosphatidyl ethanol
: amine, 5 ~ cholesterol and 5 % phosphatidinic
acid).
~ The ready-to-use reagent is prepared by
;~ reconstitution with 2 ml of aqua dest.
. ,
(concentration of the phospholipids: 65 ~g/ml~.
:: ;`
~ '
~ - 15 -
.




~' . : ,
.

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Reagent B: like reagent A, yet only 0.0017 % sulfatide.



Reagent C: 0.5 % kaolin, lipid extract fro~ porcine
mucosa (Tachostyptan~, of Hormonchemie).,
The ready-to-use reagent is prepared by
reconstitution with 2 ml of aqua dest.



Reagent D: Aktin FS~, of Baxter (contains ellagic
acid as the initiator and phospholipids from soy
bean).



Reagent E: Pathromtin~, of Behring (contains 0.5 ~
kaolin in liquid suspension as the initiator and
lipid extract from human placenta (lyo)).
The ready-to-use reagent is prepared by dissolving
the lipid extract in the kaolin suspension.



A normal plasma (NP; Immunocontrolplasma Normal,
of Immuno) or heparini~ed normal plasma containing 0.4
U heparin/ml (HPl) or 1.0 U heparin/ml (HP2) were used
as the plasma samples. 0.1 ml plasma sample was admixed
with 0.1 ml reagent. After an incubation time of two

minutes at 37C, 0.1 ml 0.025 M CaC12 solution was
admixed, and the time up to the point of coagulation
was measured with a Schnitger-Gross coagulometer (of
Amelung~.

~ .
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- 16 -


- .

,
.

Table 6 shows the results obtained, line 1
indicating the coagulation time (in seconds) of normal
plasma (NP), lines 2 and 3 indicating the coagulation
times of heparinized plasmas (HP1 and HP~), and lines 4
and 5 indicating the ratio between the coagulation
times of the heparinized plasmas and of normal plasma.
Table 6



7 Reagent C Reagent D Reagent E
w.o_pQlyh~ene 25mg/l P. ~.o.poly}~rene 25mg/l P. 7.o.Eolyb~ene 25mg/l P. ~1.o.polybrene SOmgl~P

NPsec. 34.0 32.4 52.4 68.0 43.1 65.9 34 1 46.6
HP1 sec. 52.9 36.1 123.1 75.8 99.8 67.6 76.1 50.g
¦HP2 sec. 214.1 39.1 253.4 80.3 283.4 59.1 210.1 96.2

Igatio HP1/NP 1.56 1.11 2.35 1.11 2.32 1.03 2.23 1.09
.. ¦~atio HP2/NP 1 6.30 1.21 4.84 1.18 6.58 0.90 6.16 2.06
w.o . = w ithout P. = polybrene




The results clearly indicate that in heparinized
plasmas without the presence of polybrene the aPTT is
clearly increased and thus falsified or distorted. In
the presence of polybrene, also the aPTT of heparinized
plasmas could be assayed with all the reagents tested.
The ratio between the coagulation times of the
heparinized plasmas and of normal plasma, which ideally
equals 1 (i.e., the coagulation times are exactly the

same) deviates considerably from the ideal value of 1



:-
- 17 -
~;



,
,
:. .'

~J ~

in the tests with the reagents without polybrene,
whereas the ratio of the coagulation -times in the
presence of polybrene is close to 1.
6. Factor VIII-calibration cu:rve with PTT~reagents
of diferent compositions with and without -the addition
of polybrene.
To draw these calibration curves, a reference
plasma (Reference Plasma 100 ~ of :Immuno) was used.
This Reference Plasma was dissolved in 1 ml of aqua
dest. (RP) and in 1 ml of aqua des-t. containing 1 U/ml
heparin (of Immuno) (HP).
The plasmas were pre-diluted 1:5 with imidazol
buffer (3.4 g/l imidazol, 5.85 g/l NaCl, pH 7.4); this
corresponds to 100 % of Factor VIII. Further dilutions
were prepared according to a geometric dllution series
(1:1 = 100 % to 1:128 = 0.78 %). 0.1 ml sample were
mixed with 0.1 ml reagent and incubated for 4 min at
37~C. Subsequently, 0.1 ml 0.025 M CaCl2 solution was
admixed, and the time up to the coagulation point was
measured with a Schnitger-Gross-coagulometer.
The results are indicated in Tables 7 to 10.




- 18 -
:




-~ :
- .

2~

j Table 7

Reageni A
=- __ __
,~J. o. Polybrene 25 mg/ml P~ che
F.VIII (%) RP HP RP HP
100 54.7 82.0 40 0 42.2
S0 65.2 78.6 48.6 47.6
73 5 82.1 52.0 52.1
12.5 81.7 86.6 58.3 58.0
: 6.25 90.7 92.6 64.5 64.6
- ~ 3.13 98.1 104.1 70.6 71.6
1.56 107.2 108.6 78.6 78.6
0.78 115.1 117.6 83.6 83.6
_


Table 8
~ . .

Reagent C
r----------~ ~ o PolybtenC 1 Z S mg/ml F~r~
F.VIII (%) RP HP RP
5000 891 63 12095 6 999 6 979.66
104.5 102.1 113.1 110.6
12.5 110.6 109.1 123 1 127.6
6.25 121.5 120.1 138.1 134.6
141.6 143 1 j 150.1 151.1

Table 9

Reagent D
_ == ~J.o Polybrene 25 mg/ml Po~;~b~ i
:.: F.VIII (%) ~P HP RP HP
100 60.6 107.2 70.1 65.6
S0 79 o 82 9 100.1 93.6

3 13 109.1 109 6 140.6 138.6
. ~ ¦ 0 79 118 1 118 2 164 160.5



- 1 9 -



' "
:' `'
.

?~ b~

Table 10

Reagent E
__ ~.o. ~olyb;~n~ S0 mg/ml P~
F.VIII (%) RP --HP RP HP
100 54.9 127.S 72.1 68.1
61.2 98.5 78.4 75.1
2S 67.4 96.4 87.9 82 6
: 12 S 74.Z 102.4 94.9 92.1
6.2S 83.4 112.6 106.2 10S.1
3.13 89,9 120.6 113.2 115.6
1.56 97.1 131.6 121.1 12S.S
~ 0.78 102.4 140.9 ~ 127.6 131.6
:~
~ ~ .
The results indicate that in the normal case
(plasma without heparin; RP, reagents without
polybrene) there clearly exists a correlation between
the coagulation time and the factor VIII concentration:
; the lower the concentration of factor VIII, the longer
the coagulation time.
When measuring heparin-containing samples (HP,
reagents without polybrene), no clear correlation
appears, the measurement gives a useless factor VIII-
calibration curve which does not allow for a linear
attributing of coagulation time and factor VIII
concentration~
Only the presence of polybrene in the reagents
allows for a determination of the factor VIII
concentration in heparin-containing samples (HP,
::

- reagents with 12.5/25/5Q mg/ml polybrene). The results
. .,
;~;` of the measurements are not falsified by the presence


~. of heparin and also of polybrene when using the reagent
:
~ - 20 -
:'~
;''

.. ~ : , : .

; '

` . ~ ' , .: :': ' . . ~: , .,

2 ~ ~

according to the invention. This becomes apparent both
when comparing the measurements of RP with reagents
with and without polybrene, and when comparing the
coagulation times of RP and HP in the presencé,of
polybrene, whose quotient always is quite close to l.
7. aPTT assaying with reagent B with and without
addition of heparinase.
Immunocontrolplasma Normal (oE Immuno) was
dissolved in l ml of aqua dest. containing 0, 0.5 or 1
U/ml heparin.
Reagent B was dissolved in 1 ml of aqua dest.
containing 0, 0.74, 0.22, 0.07 and 0.02 U/ml heparinase
(of IBEX, Canada).
The PTT assay was carried out as described in
Example S.
The results of the measurements carried out are
indicated in Table 11.
Table ll



heparin in plasma j heparinase U/ml I
U/ml 0 _0.02 0.07 0.22 0.74

'! . I 0.0 33.6 33.6 33.1 33.6 38.3
0.5 52.4 45.8 41.8 37.7 41.4
1.0 85.8 65.6 50.8 47.3 42.3
, __-
,
These results show that also a heparinase-
containing reagent is excellently suited for -the aPTT
assay of heparinized plasma.



- 21 -

;,.
, . ' , ' .

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(22) Filed 1993-05-13
(41) Open to Public Inspection 1993-11-16
Dead Application 1998-05-13

Abandonment History

Abandonment Date Reason Reinstatement Date
1997-05-13 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1993-05-13
Registration of a document - section 124 $0.00 1993-10-22
Maintenance Fee - Application - New Act 2 1995-05-15 $100.00 1995-03-24
Maintenance Fee - Application - New Act 3 1996-05-13 $100.00 1996-03-11
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
IMMUNO AKTIENGESELLSCHAFT
Past Owners on Record
LANG, HARTMUT
MORITZ, BERTA
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Description 
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Cover Page 1993-11-16 1 27
Abstract 1993-11-16 1 30
Claims 1993-11-16 7 193
Drawings 1993-11-16 1 13
Description 1993-11-16 20 675
Fees 1996-03-11 1 83
Fees 1995-03-24 1 89