CA2265270C - Liquid prothrombin time reagent - Google Patents
Liquid prothrombin time reagent Download PDFInfo
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- CA2265270C CA2265270C CA002265270A CA2265270A CA2265270C CA 2265270 C CA2265270 C CA 2265270C CA 002265270 A CA002265270 A CA 002265270A CA 2265270 A CA2265270 A CA 2265270A CA 2265270 C CA2265270 C CA 2265270C
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- reagent
- tissue factor
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- prothrombin time
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Liquid prothrombin time (PT) reagents for use in clotting tests, which are stable liquids that also exhibit high sensitivity and good reproducibility are disclosed. The PT reagents of the present invention comprise a recombinant tissue factor, at least one antioxidant and a serum albumin.
Description
LIQUID PROTHROMBIN TIME REAGENT
The present invention relates to a ready-to-use prothrombin time reagent which is stable long-term, based on recombinant tissue factor, and its use in clotting tests.
The prothrombin time (PT) is the most customary screening test in the field of clotting diagnosis. In this test, patient plasma is mixed with a reagent which contains at least tissue factor, phospholipids and calcium. The tissue factor can be isolated from tissue or prepared by recombinant means (Hoppenstaedt, D.A. et al. (1995) Lab. Med. 26(3), 198-203); this protein activates the extrinsic reaction pathway of clotting.
The clotting time measured depends on the concentration of the clotting factors II, V, VII and X. The evaluation of the PT is carried out in seconds of clotting time or in International Standardized Ratios (ISR). The ISR is calculated as PRISZ, where the prothrombin ratio PR is the quotient of the clotting time of the sample and a mean normal clotting time, and the International Sensitivity Index ISI is a constant which depends on the reagent and on the measuring apparatus used. The sensitivity of a reagent is all the greater, the smaller the ISI is numerically. An ISI
between 0.9 and 1.2 results in an optimum sensitivity for disorders of the extrinsic system. Even smaller ISI
values lead to extremely long clotting times in the pathological field, which for many measuring systems are no longer readily measurable.
The PT is employed:
= as a screening test for the extrinsic clotting system = for checking oral anticoagulation = for the diagnosis of liver disorders.
Customarily, most PT reagents which are available are supplied in freeze-dried form and reconstituted before use using a reconstitution medium (as a rule distilled water or a saline solution). The reason for this is the lack of stability of the reagents in the liquid state.
Adam & Eberhard (US 3,522,148) describe a liquid thromboplastin reagent based on rabbit brain thromboplastin. No statements are made about the sensitivity of the reagent.
Butler et al. (US 5,385,853) describe the preparation of a PT reagent from rabbit brain thromboplastin.
According to the invention, polyethylene glycol (PEG) is added and calcium gluconate is used as a calcium source and gentamycin as an antimicrobial agent. The ISI value achieved, however, is very high at, on average, 2.0; this corresponds to an inadequately low sensitivity of the reagent.
Two liquid thromboplastin reagents are available on the market, one from Pacific Hemostasis (USA), the other from Diamed (Basle, Switzerland). Both reagents are based on rabbit brain thromboplastin, exhibit a low sensitivity (ISI > 1.6) and have a life of 1 month and 1 year respectively.
Three different recombinant PT reagents are on the market. All are highly sensitive (ISI about 1.0) and are supplied in freeze-dried form.
The following processes have thus already been described:
= conventional freeze-dried thromboplastin reagents = conventional ready-to-use rabbit brain thromboplastin reagents with low sensitivity = recombinant freeze-dried thromboplastin reagents with high sensitivity.
Using the processes known to date, it has not been possible until now to stabilize recombinant thromboplastin such that it can be employed in a liquid reagent.
The object on which the present invention is based therefore consists in making available a liquid formulation of a recombinant thromboplastin reagent which is stable long-term. Such aveagent is extremely advantageous for the clotting laboratory, since it combines the advantages of easy handleability (ready-to-use state) with the advantages of high sensitivity and good reproducibility.
Surprisingly, it has been found that a combination of an antioxidant in a high concentration with a serum albumin in a likewise high concentration is successful, whereas the individual stabilizers are not sufficiently effective. The combination of ascorbic acid with human serum albumin, in each case in millimolar concentration, is particularly advantageous. A lower concentration of one of the two substances led to a lower stability in each case.
The stability at about 4 C is crucial for the utilizability of such a reagent. A stability of at least 12 months at 4 C is advantageous, a stability of at least 18 months is particularly advantageous and a stability of at least 24 months is very particularly advantageous.
Stability is defined here as the time constancy of the measured result for the determination of the PT of a defined plasma, e.g. of a normal plasma. The measured result counts as constant if it decreases less than 10%, preferably less than 5%.
In order to obtain rapid information about the stability at 4 C, it proves to be more favorable in practice to first subject the reagents to stress loading at 37 C.
The stability of a biological material can be estimated from these loading experiments. The equation:
(1) At = Ao e-kt applies where At = activity at the time t, Ao = activity at the start, t = time and k = degradation rate constant. The constant k is dependent on the temperature. The Arrhenius equation:
(2) log k = const 1 + [const 2/T]
approximately applies where T = absolute temperature.
In practice, the stability, for example, at 4 C is estimated by a loading experiment, for example at 37 C.
For example, a preparation whose activity has fallen by less than 5% counts as stable. For the corresponding period, after transformation of equation (1) in each case:
(3) ln (At/Ao) = ln 0.95 = -k t = -k370t370 = -k4at4o or:
(4) t4o/t37o = k37o/k4o The life at a specific temperature is thus inversely proportional to the constant k at this temperature.
The present invention relates to a ready-to-use prothrombin time reagent which is stable long-term, based on recombinant tissue factor, and its use in clotting tests.
The prothrombin time (PT) is the most customary screening test in the field of clotting diagnosis. In this test, patient plasma is mixed with a reagent which contains at least tissue factor, phospholipids and calcium. The tissue factor can be isolated from tissue or prepared by recombinant means (Hoppenstaedt, D.A. et al. (1995) Lab. Med. 26(3), 198-203); this protein activates the extrinsic reaction pathway of clotting.
The clotting time measured depends on the concentration of the clotting factors II, V, VII and X. The evaluation of the PT is carried out in seconds of clotting time or in International Standardized Ratios (ISR). The ISR is calculated as PRISZ, where the prothrombin ratio PR is the quotient of the clotting time of the sample and a mean normal clotting time, and the International Sensitivity Index ISI is a constant which depends on the reagent and on the measuring apparatus used. The sensitivity of a reagent is all the greater, the smaller the ISI is numerically. An ISI
between 0.9 and 1.2 results in an optimum sensitivity for disorders of the extrinsic system. Even smaller ISI
values lead to extremely long clotting times in the pathological field, which for many measuring systems are no longer readily measurable.
The PT is employed:
= as a screening test for the extrinsic clotting system = for checking oral anticoagulation = for the diagnosis of liver disorders.
Customarily, most PT reagents which are available are supplied in freeze-dried form and reconstituted before use using a reconstitution medium (as a rule distilled water or a saline solution). The reason for this is the lack of stability of the reagents in the liquid state.
Adam & Eberhard (US 3,522,148) describe a liquid thromboplastin reagent based on rabbit brain thromboplastin. No statements are made about the sensitivity of the reagent.
Butler et al. (US 5,385,853) describe the preparation of a PT reagent from rabbit brain thromboplastin.
According to the invention, polyethylene glycol (PEG) is added and calcium gluconate is used as a calcium source and gentamycin as an antimicrobial agent. The ISI value achieved, however, is very high at, on average, 2.0; this corresponds to an inadequately low sensitivity of the reagent.
Two liquid thromboplastin reagents are available on the market, one from Pacific Hemostasis (USA), the other from Diamed (Basle, Switzerland). Both reagents are based on rabbit brain thromboplastin, exhibit a low sensitivity (ISI > 1.6) and have a life of 1 month and 1 year respectively.
Three different recombinant PT reagents are on the market. All are highly sensitive (ISI about 1.0) and are supplied in freeze-dried form.
The following processes have thus already been described:
= conventional freeze-dried thromboplastin reagents = conventional ready-to-use rabbit brain thromboplastin reagents with low sensitivity = recombinant freeze-dried thromboplastin reagents with high sensitivity.
Using the processes known to date, it has not been possible until now to stabilize recombinant thromboplastin such that it can be employed in a liquid reagent.
The object on which the present invention is based therefore consists in making available a liquid formulation of a recombinant thromboplastin reagent which is stable long-term. Such aveagent is extremely advantageous for the clotting laboratory, since it combines the advantages of easy handleability (ready-to-use state) with the advantages of high sensitivity and good reproducibility.
Surprisingly, it has been found that a combination of an antioxidant in a high concentration with a serum albumin in a likewise high concentration is successful, whereas the individual stabilizers are not sufficiently effective. The combination of ascorbic acid with human serum albumin, in each case in millimolar concentration, is particularly advantageous. A lower concentration of one of the two substances led to a lower stability in each case.
The stability at about 4 C is crucial for the utilizability of such a reagent. A stability of at least 12 months at 4 C is advantageous, a stability of at least 18 months is particularly advantageous and a stability of at least 24 months is very particularly advantageous.
Stability is defined here as the time constancy of the measured result for the determination of the PT of a defined plasma, e.g. of a normal plasma. The measured result counts as constant if it decreases less than 10%, preferably less than 5%.
In order to obtain rapid information about the stability at 4 C, it proves to be more favorable in practice to first subject the reagents to stress loading at 37 C.
The stability of a biological material can be estimated from these loading experiments. The equation:
(1) At = Ao e-kt applies where At = activity at the time t, Ao = activity at the start, t = time and k = degradation rate constant. The constant k is dependent on the temperature. The Arrhenius equation:
(2) log k = const 1 + [const 2/T]
approximately applies where T = absolute temperature.
In practice, the stability, for example, at 4 C is estimated by a loading experiment, for example at 37 C.
For example, a preparation whose activity has fallen by less than 5% counts as stable. For the corresponding period, after transformation of equation (1) in each case:
(3) ln (At/Ao) = ln 0.95 = -k t = -k370t370 = -k4at4o or:
(4) t4o/t37o = k37o/k4o The life at a specific temperature is thus inversely proportional to the constant k at this temperature.
For the relative size of the rate constants at various temperatures, the rule of thumb has proven that an increase in the temperature by 100 leads to a doubling of the rate constants. Comparative investigations on the stability of liquid thromboplastins at various temperatures are still not available. In a comparative case (liquid clotting factor IX), observations of the degradation rate constants at various temperatures were made by Kirkwood (Kirkwood, T.B.L. (1995) Biometrics 33, 736-742). He found the following constants:
Temperature k [10-3 month-1]
-20 C 0.3 + 4 C 1.0 +20 C 7.4 +37 C 30.2 In this example, the ratio of the constants k370/k4 is thus about 30. This means that the stability of the preparation at 4 C is thirty times greater than the stability at 37 C. This means that the stability determined at 37 C in days corresponds to the stability at 4 C in months.
Loading data at 37 C are available for thromboplastin time reagents prepared according to the invention (see Example 3). If loading took place at 37 C, no fall in the activity could be detected over a period of 30 days. According to the preceding considerations, the stability of the preparation at 4 C can therefore be estimated as at least 30 months.
However, not only the stability, but in principle also the sensitivity, is crucial for a PT reagent. It is undesirable that an optionally high stability has to be bought at the expense of a loss in sensitivity. In the case of the process according to the invention, it was possible to set an ISI value of about 1.0, which is = - 6 -seen by experts as an expression of optimum sensitivity.
Figure 1 shows an ISR reference curve with an MNPT of 12.7 seconds and an ISI of 0.97 result;
Figure 2 is a graph showing prothrombin time of reagents 1 and 2; and Figure 3 is a graph showing prothrombin time of reagent 1.
The following examples are intended to illustrate the invention.
Example 1 Preparation of a prothrombin time reagent 1 part of 20% Triton X 100T"is mixed with 12 parts of 10% phospholipid suspension (PhospholiponR" 25 P, Nattermann, Germany) and 6 parts of purified recombinant human tissue factor from E. coli (about 2 mg/ml, Behring Diagnostics, Germany). After incubation at room temperature for two hours for relipidation, the batch is diluted with a 500-fold excess of buffer. The buffer consists of 50 mM HEPES
pH 7.0, 100 mM glycine and 13 mM calcium chloride, and furthermore contains the following stabilizers:
2 mM ascorbic acid 1% mannitol 1% human serum albumin (Behring Diagnostics, Germany) Example 2 ISR reference curve of a prothrombin time reagent A reagent according to Example 1 was prepared. The clotting times for the AK calibration plasmas (Immuno, Austria) were determined on a Behring coagulation timer (Behring Diagnostics). These are lyophilized Marcumar plasmas having declared ISR values.
MNPT and ISI can be estimated from the data (see Fig. 1) Example 3 Stability of a prothronnbin time reagent Reagent 1 was prepared according to Example 1.
Differing from Example 1, reagent 2 only contains 0.1%
human serum albumin. The ISI was determined according to Example 2.
Reagents were loaded at 37 C. Samples were taken in the course of time and the prothrombin time of lyophilized normal plasma and lyophilized pathological plasma (standard human plasma and PathoplasmaT" II, Behring Diagnostics) were determined on a Behring coagulation timer (see Fig. 2).
Temperature k [10-3 month-1]
-20 C 0.3 + 4 C 1.0 +20 C 7.4 +37 C 30.2 In this example, the ratio of the constants k370/k4 is thus about 30. This means that the stability of the preparation at 4 C is thirty times greater than the stability at 37 C. This means that the stability determined at 37 C in days corresponds to the stability at 4 C in months.
Loading data at 37 C are available for thromboplastin time reagents prepared according to the invention (see Example 3). If loading took place at 37 C, no fall in the activity could be detected over a period of 30 days. According to the preceding considerations, the stability of the preparation at 4 C can therefore be estimated as at least 30 months.
However, not only the stability, but in principle also the sensitivity, is crucial for a PT reagent. It is undesirable that an optionally high stability has to be bought at the expense of a loss in sensitivity. In the case of the process according to the invention, it was possible to set an ISI value of about 1.0, which is = - 6 -seen by experts as an expression of optimum sensitivity.
Figure 1 shows an ISR reference curve with an MNPT of 12.7 seconds and an ISI of 0.97 result;
Figure 2 is a graph showing prothrombin time of reagents 1 and 2; and Figure 3 is a graph showing prothrombin time of reagent 1.
The following examples are intended to illustrate the invention.
Example 1 Preparation of a prothrombin time reagent 1 part of 20% Triton X 100T"is mixed with 12 parts of 10% phospholipid suspension (PhospholiponR" 25 P, Nattermann, Germany) and 6 parts of purified recombinant human tissue factor from E. coli (about 2 mg/ml, Behring Diagnostics, Germany). After incubation at room temperature for two hours for relipidation, the batch is diluted with a 500-fold excess of buffer. The buffer consists of 50 mM HEPES
pH 7.0, 100 mM glycine and 13 mM calcium chloride, and furthermore contains the following stabilizers:
2 mM ascorbic acid 1% mannitol 1% human serum albumin (Behring Diagnostics, Germany) Example 2 ISR reference curve of a prothrombin time reagent A reagent according to Example 1 was prepared. The clotting times for the AK calibration plasmas (Immuno, Austria) were determined on a Behring coagulation timer (Behring Diagnostics). These are lyophilized Marcumar plasmas having declared ISR values.
MNPT and ISI can be estimated from the data (see Fig. 1) Example 3 Stability of a prothronnbin time reagent Reagent 1 was prepared according to Example 1.
Differing from Example 1, reagent 2 only contains 0.1%
human serum albumin. The ISI was determined according to Example 2.
Reagents were loaded at 37 C. Samples were taken in the course of time and the prothrombin time of lyophilized normal plasma and lyophilized pathological plasma (standard human plasma and PathoplasmaT" II, Behring Diagnostics) were determined on a Behring coagulation timer (see Fig. 2).
Claims (10)
1. A liquid prothrombin time reagent comprising recombinant tissue factor, at least one antioxidant and a serum albumin.
2. The reagent as claimed in claim 1, wherein ascorbic acid is used as an antioxidant.
3. The reagent as claimed in claim 2, wherein ascorbic acid is used in a concentration of 1 µM to 0.1 M.
4. The reagent as claimed in claim 1, wherein the serum albumin is of human, animal or recombinant origin.
5. The reagent as claimed in claim 4, wherein the serum albumin is of human origin.
6. The reagent as claimed in claim 5, wherein the human serum albumin is added in a concentration of 0.1 g/l to 100 g/l.
7. The reagent as claimed in claim 1, wherein the recombinant tissue factor used is of human or animal origin.
8. The reagent as claimed in claim 7, wherein the recombinant tissue factor is a human recombinant tissue factor.
9. The reagent as claimed in claim 7, wherein the tissue factor is cloned and expressed in cells of human, animal, vegetable, fungal or microbial origin.
10. The reagent as claimed in claim 9, wherein the tissue factor is cloned and expressed in Escherichia coli.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19811016.2 | 1998-03-13 | ||
| DE19811016A DE19811016A1 (en) | 1998-03-13 | 1998-03-13 | Ready-to-use prothrombin time reagent based on recombinant tissue factor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2265270A1 CA2265270A1 (en) | 1999-09-13 |
| CA2265270C true CA2265270C (en) | 2009-11-24 |
Family
ID=7860840
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002263197A Withdrawn CA2263197A1 (en) | 1998-03-13 | 1999-03-01 | Ready-to-use prothrombin time reagent based on recombinant tissue factor |
| CA002265270A Expired - Lifetime CA2265270C (en) | 1998-03-13 | 1999-03-11 | Liquid prothrombin time reagent |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002263197A Withdrawn CA2263197A1 (en) | 1998-03-13 | 1999-03-01 | Ready-to-use prothrombin time reagent based on recombinant tissue factor |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0942284B1 (en) |
| JP (1) | JP4199365B2 (en) |
| AT (1) | ATE385315T1 (en) |
| CA (2) | CA2263197A1 (en) |
| DE (2) | DE19811016A1 (en) |
| ES (1) | ES2299224T3 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7049087B2 (en) * | 2002-11-05 | 2006-05-23 | Lifescan, Inc. | Method for manufacturing a tissue factor-based prothrombin time reagent |
| JP4377207B2 (en) * | 2003-11-28 | 2009-12-02 | シスメックス株式会社 | Blood coagulation time measurement method and blood coagulation time measurement reagent |
| US7148067B2 (en) * | 2004-08-31 | 2006-12-12 | The Board Of Trustees Of The University Of Illinois | Thromboplastin reagents |
| DE602006021290D1 (en) | 2005-03-04 | 2011-05-26 | Univ Illinois | MODULATOR OF COAGULATION CASCADES AND FIBRINOLYTIC CASCADES |
| WO2009046194A2 (en) | 2007-10-05 | 2009-04-09 | The Board Of Trustees Of The University Of Illinois | Fibrin sealant |
| WO2009061697A1 (en) | 2007-11-09 | 2009-05-14 | The Board Of Trustees Of The University Of Illinois | Anticoagulant antagonist and hemophilia procoagulant |
| DE102007062323A1 (en) * | 2007-12-21 | 2009-06-25 | Siemens Healthcare Diagnostics Products Gmbh | Long-term stable thromboplastin reagent |
| PL2528617T3 (en) | 2010-01-28 | 2016-09-30 | Interleukin-2 or interferon-alpha for use in treating nicotine or food addiction | |
| JP6116231B2 (en) * | 2012-12-25 | 2017-04-19 | 株式会社Ｌｓｉメディエンス | Tissue thromboplastin-containing blood coagulation ability measuring reagent and measuring method |
| EP2772762B1 (en) * | 2013-02-28 | 2016-11-30 | Siemens Healthcare Diagnostics Products GmbH | Method for determining protein S activity |
| JP6250306B2 (en) * | 2013-05-28 | 2017-12-20 | 株式会社Ｌｓｉメディエンス | Respirated thromboplastin-containing blood coagulation ability measuring reagent and measuring method |
| AU2017217804B2 (en) | 2016-02-12 | 2020-10-22 | Instrumentation Laboratory Company | A two component "mix and use" liquid thromboplastin reagent, methods of making, and methods of use thereof |
| EP3489692A1 (en) | 2017-11-28 | 2019-05-29 | Siemens Healthcare Diagnostics Products GmbH | Prothrombin time reagent comprising an iron chelator |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3522148A (en) * | 1965-08-13 | 1970-07-28 | Dade Reagents Inc | Stabilized thromboplastin preparation |
| US5314695A (en) * | 1990-11-13 | 1994-05-24 | Corvas International, Inc. | Tissue factor based prothrombin time reagent |
| US5358853A (en) * | 1992-08-03 | 1994-10-25 | Akzo Av | Liquid thromboplastin reagent |
| CA2288058A1 (en) * | 1997-04-23 | 1998-10-29 | Instrumentation Laboratory, S.P.A. | Recombinant rabbit tissue factor based prothrombin time reagent |
-
1998
- 1998-03-13 DE DE19811016A patent/DE19811016A1/en not_active Withdrawn
-
1999
- 1999-02-12 AT AT99102672T patent/ATE385315T1/en not_active IP Right Cessation
- 1999-02-12 ES ES99102672T patent/ES2299224T3/en not_active Expired - Lifetime
- 1999-02-12 DE DE59914634T patent/DE59914634D1/en not_active Expired - Lifetime
- 1999-02-12 EP EP99102672A patent/EP0942284B1/en not_active Expired - Lifetime
- 1999-03-01 CA CA002263197A patent/CA2263197A1/en not_active Withdrawn
- 1999-03-11 CA CA002265270A patent/CA2265270C/en not_active Expired - Lifetime
- 1999-03-12 JP JP06585699A patent/JP4199365B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP4199365B2 (en) | 2008-12-17 |
| CA2265270A1 (en) | 1999-09-13 |
| DE19811016A1 (en) | 1999-09-16 |
| DE59914634D1 (en) | 2008-03-20 |
| CA2263197A1 (en) | 1999-09-13 |
| ATE385315T1 (en) | 2008-02-15 |
| EP0942284A2 (en) | 1999-09-15 |
| EP0942284B1 (en) | 2008-01-30 |
| ES2299224T3 (en) | 2008-05-16 |
| JPH11316228A (en) | 1999-11-16 |
| EP0942284A3 (en) | 2005-03-16 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20190311 |