PIPETTE TIP AND USES AND METHODS THEREOF

Abstract

The present invention provides a pipette tip, which can be used in in-vitro diagnostics, in particular in the diagnostic testing of body fluids, such as in coagulation testing. The Pipette tip contains two constituents in a spatially separated manner. The present invention furthermore provides a method of performing such diagnostics, e.g. coagulation analysis, and to the use of the pipette tip in such diagnostic testing.

Claims

1. A pipette tip (11) comprising a constituent (A) and a constituent (B) in a spatially separated manner, wherein the constituents (A) and (B) are adapted to form a diagnostic composition upon combination.

2. The pipette tip according to claim 1, wherein the diagnostic composition comprises at least the following components (i) and (ii): i) an activator of coagulation; and ii) a calcium salt.

3. The pipette tip according to claim 1 or 2, wherein the pipette tip comprises a) a compartment (a) containing the constituent (A); and/or b) a compartment (b) containing the constituent (B).

4. The pipette tip according to claim 3, wherein: a) the compartment (a) containing the constituent (A) does not contain the constituent (B); and/or b) the compartment (b) containing the constituent (B) does not contain the constituent (A).

5. The pipette tip according to any of claims 1-4, wherein the constituent (A) is different from the constituent (B).

6. The pipette tip according to any of claims 1 to 5, wherein each of the constituents (A) and (B) is independently from each other a liquid formulation, an essentially dry formulation, or a dry formulation.

7. The pipette tip according to claim 6, wherein constituents (A) and (B) are essentially dry formulations or dry formulations, preferably constituents (A) and (B) are dry formulations.

8. The pipette tip according to any of claims 2 to 7, wherein 1) constituent (A) comprises component (i) but not component (ii) and constituent (B) comprises component (ii) but not component (i); or 2) constituent (A) comprises component (ii) but not component (i) and constituent (B) comprises component (i) but not component (ii).

9. The pipette tip according to any of claims 2 to 8, wherein the activator of coagulation is an extrinsic activator and/or an intrinsic activator.

10. The pipette tip according to any of claims 2 to 9, wherein component (i) is an extrinsic activator of coagulation and component (ii) is a calcium salt; or wherein component (i) is an intrinsic activator of coagulation and component (ii) is a calcium salt.

11. The pipette tip according to claim 9 or 10, wherein the extrinsic activator is a Tissue Factor (TF), which is preferably selected from lipidated TF or rTF.

12. The pipette tip according to claim 9 or 10, wherein the intrinsic activator of coagulation is selected from the group consisting of celite, ellagic acid, sulfatit, kaolin, silica, RNA, and mixtures thereof.

13. The pipette tip according to any of claims 2 to 12, wherein the calcium salt is calcium chloride and/or calcium lactate and/or calcium gluconate.

14. The pipette tip according to any of claims 1 to 13, wherein the diagnostic composition comprises one or more components selected from the group consisting of a coagulating activating factor, a coagulation inhibitor and an active-component inhibitor.

15. The pipette tip according to claim 14, wherein the active-component inhibitor is selected from one or more platelet inhibitors, fibrinolysis inhibitors, and/or heparin inhibitors.

16. The pipette tip according to claim 15, wherein the platelet inhibitor is a cytoskeleton inhibitor or a GPIIb/IIIa antagonist.

17. The pipette tip according to claim 15, wherein the fibrinolysis inhibitor is selected from the group consisting of aprotinin, tranexamic acid, eaca, thrombin-activated fibrinolysis inhibitor, plasminogen activation inhibitor 1/2, ?2-antiplasmin, and ?2-macroglobulin.

18. The pipette tip according to claim 15, wherein the heparin inhibitor is selected from heparinase, protamine or protamine-related peptides and their derivatives, or other cationic polymers, for example hexadimethrine bromide (polybrene).

19. The pipette tip according to any of claims 14 to 18, wherein the coagulation activating factor is selected from the group consisting of FI, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, and TF.

20. The pipette tip according to any of claims 14 to 19, wherein the coagulation inhibitor is selected from tissue factor pathway inhibitor, antithrombin I-IV, or activated protein C.

21. The pipette tip according to any of claims 3 to 20, wherein the phospholipid and/or the heparin inhibitor, preferably heparinase and/or hexadimethrine bromide (polybrene), more preferably hexadimethrine bromide (polybrene), are comprised by constituent (A) and arranged in compartment (a).

22. The pipette tip according to any of claims 1 to 21, wherein a protein stabilizer, for example albumin or gelatin, is comprised by constituent (A) but is not comprised by constituent (B); or wherein a protein stabilizer, for example albumin or gelatin, is comprised by constituent (B) but is not comprised by constituent (A).

23. The pipette tip according to any of claims 1 to 22, wherein the pipette tip comprises at least one porous insert (14), preferably the porous insert having a minimum pore diameter of 2 ?m and a maximum pore diameter of 2.0 mm and preferably containing the constituent (A) or the constituent (B).

24. The pipette tip according to claim 23, wherein the shape of the pipette tip (11) is adapted to receive a porous insert (14) in its lower part, whereby the porous insert (14) preferably has a cylindrical shape.

25. The pipette tip according to any of claims 1 to 24, wherein the pipette tip (11) comprises at least one reagent layer (15), the reagent layer (15) preferably located at the lowest part of the pipette tip (11) and having an even thickness.

26. The pipette tip according to any of claims 1 to 25, wherein the pipette tip (11) comprises a reagent layer (15) that is formed by spraying of drops into the pipette tip, whereby each of the sprayed drops has a diameter of less than 100 ?m.

27. The pipette tip according to claim 25 or 26, wherein the pipette tip (11) comprises at least one porous insert (14).

28. The pipette tip according to claim 27, wherein the porous insert (14) comprises constituent (A) and the reagent layer (15) comprises constituent (B) or the porous insert (14) comprises constituent (B) and the reagent layer (15) comprises constituent (A).

29. The pipette tip according to claim 27 or 28, wherein the porous insert (14) is located above the at least one reagent layer (15) in the pipette tip.

30. The pipette tip according to any of claims 1 to 29, wherein the pipette tip (11) is a disposable pipette tip.

31. Use of the pipette tip according to any of claims 1 to 30 in a coagulation test.

32. Use according to claim 31 in a viscoelastic analysis of a sample.

33. A method of performing a diagnostic test on a sample comprising the following steps: (1) providing a pipette tip (11) according to any of claims 1-30; (2) aspirating the sample into the pipette tip, thereby mixing, preferably dissolving, the constituent (A), e.g. contained in compartment (a), and the constituent (B), e.g. contained in compartment (b), of said pipette tip in the sample and obtaining a mixture, preferably a solution, of the sample and constituents (A) and (B), wherein in said mixture constituents (A) and (B) form a diagnostic composition that is required to perform the diagnostic test on the sample; (3) optionally, transferring the mixture of the sample and the diagnostic composition into a measurement container suitable for performing said diagnostic test, such as a cuvette; (4) optionally, putting the measurement container, such as the cuvette, into an apparatus suitable for performing said diagnostic test; and (5) performing the diagnostic test of said mixture, optionally in the measurement container, such as the cuvette.

34. The method according to claim 33, wherein the sample is a human or mammalian body fluid, preferably a human or mammalian blood sample or a fraction of a blood sample.

35. The method according to claim 33 or 34, wherein the sample comprises whole blood and/or blood plasma.

36. The method according to claim 33, wherein the diagnostic test is a coagulation test and the sample is a blood sample or a fraction of a blood sample.

37. The method of claim 36, wherein the coagulation test is viscoelastic analysis.

38. The method according to claim 36 or 37, wherein the diagnostic test in step (5) comprises the determination of the clotting time, the clot formation time, the firmness of the clot over time or the fibrinolysis activity as firmness reduction in relation to the maximum clot firmness, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0204] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0205] FIG. 1 is an exemplary diagram showing a typical viscoelastic measurement and corresponding curve parameters: clotting time CT is the lag time between activation of the sample and the time when a firmness value of 2 mm is reached; clot formation time CFT is the time that passes between the firmness values of 2 mm and 20 mm; alpha is the angle that is formed between the tangential of the firmness curve and the x-axis; maximum clot firmness MCF is the maximum firmness value of the curve; maximum lysis is the percentage decrease of firmness after MCF has been reached.

[0206] FIG. 2 shows an illustration of an apparatus for viscoelastic testing: After the formation of the clot between cup 1 (cuvette) and pin 2, the clot itself is stretched by the movement of the pin 2 relative to the cup 1. The detection of the characteristic parameters of the clot is based on the mechanical coupling of cup 1 and pin 2 by the clot. This is only possible if the clot adheres on the surfaces of both cup 1 and pin 2. Thus, a firm adhesion to the surfaces of both cup 1 and pin 2 is typically required for the viscoelastic analysis. During a viscoelastic measurement, the pin is fixed to the axis 4 and gently and slowly rotated in the cup via the spring 7. The axis 4 itself is fixed to the base plate 5 with the ball bearing 6. The movement of the pin is measured optically by illuminating the mirror 9 (fixed to the axis 4) with the light source 8 and detecting the reflected signal at the spatially resolving photo detector 10.

[0207] FIG. 3 shows schematic cross-sectional views of six preferred embodiments of a pipetting tip (11) containing constituents (A) and (B): [0208] a) longitudinal cross-section through a conventional pipette tip (11a) with open lower end (12a), open upper end (13a) fitting to the pipette dimensions, porous insert (14a) and reagent layer (15a) below the porous insert (14a); [0209] b) longitudinal cross-section through a modified pipette tip (11 b) with open lower end (12b), open upper end (13b) fitting to the pipette dimensions, porous insert (14b) and reagent layer (15b) below the porous insert (14b); [0210] c) longitudinal cross-section through a modified pipette tip shape (11c) with open lower end (12c), open upper end (13c) fitting to the pipette dimensions, and two porous inserts (14c, 14c); [0211] d) longitudinal cross-section through a conventional pipette tip (11d) with open lower end (12d), open upper end (13d) fitting to the pipette dimensions, porous insert (14d) and reagent layer (15d) above the porous insert (14d); [0212] e) longitudinal cross-section through a conventional pipette tip (11e) with open lower end (12e), open upper end (13e) fitting to the pipette dimensions, and two circumferential reagent layers (15e, 15e) located on top of each other; and [0213] f) longitudinal cross-section through a conventional pipette tip (11f) with open lower end (12f), open upper end (13f) fitting to the pipette dimensions, and two spot-like reagent layers (15f, 15f) located in juxtaposition.

LIST OF REFERENCE SIGNS

[0214] 1, 1a, 1b, 1c measurement cup

[0215] 2, 2a, 2b, 2c pin

[0216] 3 sample

[0217] 4 axis

[0218] 5 base plate

[0219] 6 ball bearing

[0220] 7 spring

[0221] 8 light source

[0222] 8 mirror

[0223] 10 detector

[0224] 11, 11a, 11b, 11c, 11d, 11e, 11f pipette tip

[0225] 12a, 12b, 12c, 12d, 12e, 12f open lower end of the pipette tip

[0226] 13a, 13b, 13c, 13d, 13e, 13f open upper end of the pipette tip

[0227] 14a, 14b, 14c, 14c, 14d porous insert

[0228] 15a, 15b, 15d, 15e, 15e, 15f, 15f reagent layer

EXAMPLES

[0229] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1

Effect of TF/Phospholipids Deposited either in Compartment (a) or in Compartment (b) of the Pipette Tip on Clotting Time

[0230] To investigate the effect of the extrinsic activator of coagulation on the clotting time when TF and phospholipids are deposited in dry form either in compartment (a) or in compartment (b), viscoelastic measurements of human plasma samples (10 donors mixed) were performed with a ROTEG? 05 device (Pentapharm GmbH, Munich, Germany). In the pipette tip used herein, compartment (b) was formed by a cylindrical porous insert made of polyether foam (RG 130 grey, Hildebrandt and Richter & Co. GmbH, Germany), the porous insert having a cylindrical shape of 5 mm height and 4 mm diameter. The porous insert was located in the lower half of the pipette tip. Compartment (a) of the pipette tip was provided by a crosswise section of the pipette tip, wherein a spot-like reagent layer of approximately 1.5 mm diameter (corresponding to about 4 it liquid reagent before drying) was deposited.

[0231] Frozen plasma samples where freshly thawed and heated to 37? C. just before measurement. The source of TF/phospholipids was Innovin? (Siemens AG, Germany) and the source of CaCl.sub.2 was Calcium Chloride Dihydrate (Sigma-Aldrich Chemie GmbH, Germany). Pipetting of TF was performed with Top-Line? 1 ml tips (AHN Biotechnologie GmbH, Germany) on a manual 1 ml pipettor (Brand, Germany). Liquid CaCl.sub.2 was placed in the measurement cuvette just before TF pipetting was performed. The TF composition contained 15 ul of Innovin? standard stock solution together with 4% sucrose and was dried for 2 days in a desiccator filled with 100 g molecular sieve (4 Angstroem) after placing either in compartment (a) or in compartment (b). For the control experiment, the same sample was measured by using the standard liquid reagent provided for the ROTEG 05 system (TEM Innovations GmbH, Germany).

[0232] Results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Clotting times (CT) obtained after placing the same amounts of TF in the pipette tip in either dry or wet form either in compartment (a) or in compartment (b). Each value was calculated as average of 4 measurements with human plasma. Mixed storage of TF and CaCl.sub.2 impairs the clotting time CT severely (correction impossible), while mixing TF with 4% sucrose can compensate for degradation during storage and/or dissolution delays of pure TF. CT of dry CT of dry CT of wet CT of wet TF in TF in TF in TF in com- com- com- com- CT partment partment partment partment Activator control (a) (b) (a) (b) Tissue factor/ 57 sec 61 sec 239 sec 55 sec 168 sec phospholipids

Example 2

Effect of TF/Phospholipids Deposited in the Pipette Tip alone or in Combination with CaCl2 on Clotting Time

[0233] To investigate the effect of the extrinsic activator of coagulation TF and phospholipids deposited in dry form in compartment (a) of the pipette tip either alone or in combination with CaCl.sub.2 on the clotting time, viscoelastic measurements of human plasma samples (10 donors mixed) were performed with a ROTEG? 05 device (Pentapharm GmbH, Munich, Germany) according to the procedures described in Example 1. Compartment (a) of the pipette tip was provided by a crosswise section in the lower third of the pipette tip, wherein a spot-like reagent layer of about 2 mm diameter, corresponding to about 5 ?l liquid reagent before drying, was deposited.

[0234] Results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Clotting times (CT) obtained after drying the same amounts of TF solution in compartment (a) of the pipette tip and storing for one week at room temperature. For the pure TF sample, the same amount of CaCl.sub.2 as in the mixed sample was added just before the measurement (each value was calculated as average of 4 measurements with human plasma). Mixed storage of TF and CaCl.sub.2 impairs the clotting time CT severely (correction impossible), while increasing the amount of TF by a factor of 4 and adding 2% sucrose can compensate for degradation of the pure TF sample during storage and/or dissolution delays. CT of TF/CaCl.sub.2 CT of CT of pure TF Activator CT control mixture pure TF (4x concentr.) Tissue factor/ 59 sec >600 sec 122 sec 57 sec phospholipids

[0235] Taken together, the results of this experiment show that TF and CaCl.sub.2 should not be stored mixed together. Thus, separation of TF and CaCl.sub.2 into two spatially separated compartments (a) and (b) seems undoubtedly necessary.

Example 3

Effect of Ellagic Acid/Phospholipids Deposited in the Pipette Tip in Wet or Dry Form on Clotting Time

[0236] To investigate the effect of an intrinsic activator of coagulation, namely ellagic acid and phospholipids, provided either in dry or in wet form in compartment (b) of the pipette tip (formed by a porous insert as described in Example 1) on clotting time, viscoelastic measurements were performed by using the equipment and procedures described in Example 1 above.

[0237] Results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Clotting times (CT) obtained by identical activator solutions without additives after storage as liquid or dried tip for 7 days at room temperature (tip insert made from polyether, each value was calculated as average of 4 measurements with human plasma). The wet storage of ellagic acid results in comparable CT values as the control, but the degradation during dry storage and/or dissolution delay can be compensated for by 35% more activator content and adding 2% sucrose. CT of CT of dry tip Activator CT control dry tip CT of wet tip (1,3x concentr.) Ellagic acid/ 164 sec 258 sec 162 sec 161 sec phospholipids

Example 4

Effect of CaCl.SUB.2 .Deposited in the Pipette Tip in either Wet or Dry Form in Compartment (a) or (b) on Clotting Time

[0238] To investigate the effect of CaCl.sub.2 deposited either in dry or in wet form in compartment (a) or (b) of the pipette tip, viscoelastic measurements were performed by using the procedures, equipment and compartment specifications as described in Example 1 above.

[0239] Results are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Clotting times (CT) obtained after storing CaCl.sub.2 in the tip for one week at room temperature (each value was calculated as average of 4 measurements with human plasma). No significant differences to the control CT are observed for all four approaches. CaCl.sub.2 CaCl.sub.2 dry in wet in CaCl.sub.2 dry in CaCl.sub.2 wet in compart- compart- CT compartment compartment ment ment Activator control (a) (a) (b) (b) Ellagic 194 sec 207 sec 205 sec 192 sec 189 sec acid