Coated articles for blood coagulation testing and methods of preparing the same

Abstract

The present invention provides a coated article, which can be used in in-vitro diagnostics, in particular in the diagnostic testing of body fluids, such as in blood coagulation testing. The coated article is made of a polymer material and coated with a polymer material, which may be the same or different. The present invention furthermore provides a method of preparing such a coated article and a method of performing such diagnostics, e.g. coagulation analysis.

Claims

1. A pair of first and second disposable articles adapted for use in a viscoelastic measurement apparatus for measuring coagulation characteristics of a blood sample, the first and second disposable articles being adapted to move with respect to each other, a volume being defined between surfaces of the first and second disposable articles, the volume adapted to receive the sample of blood, each of the first and second disposable articles comprising: (a) an article body made of a polymer material, the polymer material comprising a first polymer, and (b) a coating material layer, distinct from the article body, disposed on the surfaces defining the volume adapted to receive the sample of blood, the coating material layer comprising a coating material selected from the group consisting of a second polymer, a resin, and combinations thereof, the coating material layer being configured to adhere to a fibrin network during coagulation of the blood sample; wherein, (i) the first disposable article is a measurement cup, (ii) the second disposable article is selected from the group consisting of a pin and a sleeve for a pin, and (iii) the measurement apparatus is selected from the group consisting of a thromboelastography measurement apparatus and a thromboelastometry measurement apparatus.

2. The pair of disposable articles according to claim 1, wherein the coating material comprises at least one second polymer.

3. The pair of disposable articles according to claim 2, wherein the second polymer comprises a monomer selected from the group consisting of a styrene monomer, a (meth)acrylate monomer, a (meth)acrylamide monomer, an alkyl monomer, a vinyl monomer, an ally I monomer, a carbonate monomer, an aromatic monomer, an olefin monomer, a halogenolefine monomer, a methylolefine monomer, a urethane monomer, an amide monomer, an ester monomer, and an ether monomer.

4. The pair of disposable articles according to claim 2, wherein the second polymer is selected from the group consisting of a polystyrene, a polycarbonate, a polymethacrylate, a polyolefine, a polyhalogenolefine, a polymethylolefine, a polyacetal a polyurethane, a polyamide, a polyaramide, a polyester, a polyether, a polyketone, partially substituted polymers of any of the foregoing, and co-polymers of any of the foregoing.

5. The pair of disposable articles according to claim 2, wherein the second polymer comprises a monomer selected from the group consisting of a styrene monomer, a (meth)acrylate monomer, a (meth)acrylamide monomer, a carbonate monomer, an amide monomer, and an aromatic monomer.

6. The pair of disposable articles according to claim 2, wherein the second polymer is selected from the group consisting of acrylonitrile butadiene styrene (ABS), methyl methacrylate acrylonitrile butadiene styrene (MABS), polystyrene (PS), high impact polystyrene (HIPS), poly(methyl methacrylate) (PMMA), polycarbonate (PC), polyamide (PA), and polyphenylene sulfide (PPS).

7. The pair of disposable articles according to claim 2, wherein the first polymer is the same as the second polymer.

8. The pair of disposable articles according to claim 2, wherein the first polymer is distinct from the second polymer.

9. The pair of disposable articles according to claim 1, wherein the coating material comprises at least one resin.

10. The pair of disposable articles according to claim 9, wherein the resin is selected from the group consisting of an epoxy resin, a phenol resin, a polyurethane resin, an acrylate resin, and combinations thereof.

11. The pair of disposable articles according to claim 1, wherein the coating material further comprises a dye.

12. The pair of disposable articles according to claim 1, wherein the coating material further comprises a particle enabling determination of the quality of the coating material layer.

13. The pair of disposable articles according to claim 1, wherein the first polymer is a mass-production compatible plastic.

14. The pair of disposable articles according to claim 1, wherein the first polymer is selected from the group consisting of a thermoplastic, a thermoplastic elastomer, a conventional elastomer, and a duromer.

15. The pair of disposable articles according to claim 1, wherein the first polymer is selected from the group consisting of polymethylpentene (PMP), methyl methacrylate acrylonitrile butadiene styrene (MABS), and combinations thereof.

16. The pair of disposable articles according to claim 1, wherein the coating material layer is 100 nm to 100 μm in thickness.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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.

(2) FIG. 1: shows the pathway of blood coagulation resulting in the formation of fibrin strands after either extrinsic or intrinsic activation. The different enzymatic factors are indicated by their common short names.

(3) FIG. 2: Schematic representation of the blood-clot structure including fibrin strands (black), activated thrombocytes (light gray), and erythrocytes (dark gray).

(4) FIG. 3: Scanning electron microscopy image of coagulated blood consisting of fibrin strands, activated thrombocytes and erythrocytes. The white bar indicates a length of 5 μm.

(5) FIG. 4: shows a schematic drawing of a thromboelastometric device measuring the clot formation of coagulating blood by sensing the increasing shear modulus via a spring-driven oscillation in small angle ranges. After the formation of the clot between cup (“cuvette”) and pin (“probe”), the clot itself is stretched by the movement of the pin relative to the cup. The detection of the characteristic parameters of the clot is based on the mechanical coupling of cup and pin by the clot (fibrin strands and platelet aggregates between pin and cup surfaces). This is only possible if the clot adheres on the surfaces of both, cup and pin. Thus, a firm adhesion to the surfaces of both cup and pin is typically required for viscoelastic analysis. During a viscoelastic measurement, the pin is fixed to the rotating axis and gently and slowly rotated in the cup via the spring. The axis itself is fixed to a base plate, e.g. by a ball bearing. The movement of the pin is measured optically by illuminating a mirror (fixed to the rotating axis) by use of a light source and detecting the reflected signal at the spatially resolving photo detector.

(6) FIG. 5: Different shapes of thromboelastometric measurements indicating normal coagulation behavior and three typical disease patterns.

(7) FIG. 6: Comparison of the thromboelastric patterns in the case of normal coagulation (A), pathologic hyperfibrinolysis (B), and artificial fibrin network tear-offs (C).

(8) FIG. 7: shows for Example 2 clot firmness amplitude after 20 minutes (A20) in thromboelastometric measurements of different untreated articles (cuvettes and probes) made of polymethylpentene (PMP), methyl methacrylate acrylonitrile butadiene styrene (MABS), polyamide (PA), polyphenylene sulfide (PPS), or polyurethane (PU). The mean and standard deviation values were obtained from 8 individual measurements with one blood sample.

(9) FIG. 8: shows for Example 2 maximum lysis activity (ML, % ratio between clot firmness 60 minutes after measurement start and maximum clot firmness) in thromboelastometric measurements of different untreated articles (cuvettes and probes) made of polymethylpentene (PMP), methyl methacrylate acrylonitrile butadiene styrene (MABS), polyamide (PA), polyphenylene sulfide (PPS), or polyurethane (PU). The mean and standard deviation values were obtained from 8 individual measurements with one blood sample.

(10) FIG. 9: shows for Example 3 a typical thromboelastography trace of an untreated/uncoated article (cuvette and probe) made of Polymethylpentene (PMP) and reflecting partial ‘tear-offs’ (A) in comparison to the typical thromboelastography trace obtained with an article made of identical material (PMP), but coated with MABS (B).

(11) FIG. 10: shows for Example 4 clot firmness amplitude after 20 minutes (A20) in thromboelastometric measurements of differently treated/coated articles (cuvettes and probes) made of PMP. The mean and standard deviation values were obtained from 12 individual measurements with one blood sample.

(12) FIG. 11: shows for Example 4 maximum lysis activity (ML, % ratio between clot firmness 60 minutes after measurement start and maximum clot firmness) in thromboelastometric measurements of differently treated/coated articles (cuvettes and probes) made of PMP. The mean and standard deviation values were obtained from 12 individual measurements with one blood sample.

(13) FIG. 12: shows for Example 5 clot firmness amplitude after 20 minutes (A20) in thromboelastometric measurements of differently coated articles (cuvettes and probes) made of MABS. The mean and standard deviation values were obtained from 12 individual measurements with one blood sample.

(14) FIG. 13: shows for Example 5 maximum lysis activity (ML, % ratio between clot firmness 60 minutes after measurement start and maximum clot firmness) in thromboelastometric measurements of differently coated articles (cuvettes and probes) made of MABS. The mean and standard deviation values were obtained from 12 individual measurements with one blood sample.

EXAMPLES

(15) 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: Coating of Various Articles and Functionality Tests

(16) In the following examples, several exemplary results obtained with different articles according to the present invention (summarized, e.g., in FIG. 8-10) will be described. To obtain an article according to the present invention, an uncoated article made of a first polymer material was coated by using a coating composition as described below.

(17) In general, to obtain the coating compositions, the following solvents were used to dissolve 500 mg of each raw polymer material in 5 ml solvent (for detailed description see the examples below): Acrylonitrile butadiene styrene (ABS; Terluran GP-22, INEOS Styrolution Group GmbH, Germany) was dissolved in 96% xylene; Methyl methacrylate acrylonitrile butadiene styrene (MABS; TERLUX® 2802, INEOS Styrolution Group GmbH, Germany) was dissolved in 96% xylene or in ethylbutanol (Sigma-Aldrich Chemie GmbH, Germany) as indicated; High impact polystyrene (HIPS; Styrolution PS 495N, INEOS Styrolution Group GmbH, German)) was dissolved in 96% xylene; Poly(methyl methacrylate) (PMMA; Plexiglas®, EVONIK Industries AG, Germany) was dissolved in 96% acetone; Polycarbonate (PC; Lexan™ 144R, Germany) was dissolved in 96% chloroform; Polyamide (PA; Trogamid® T5000, Evonik Industries AG, Germany) was dissolved in 96% DMSO, but the solution was not properly applicable as coating composition due to the high polarity of DMSO; and polyurethane (PU; Desmopan® 385 S, Bayer MaterialScience AG, Germany) was dissolved in 96% DMSO, but the solution was not properly applicable as coating composition due to the high polarity of DMSO.

(18) In general, in the experiments explained in detail below, similar results were obtained for the solvents xylene, ethylbutanol, or a combination thereof (e.g., 50:50).

(19) To achieve sufficient coating of all surface areas that are in blood contact during a thromboelastographic measurement, articles to be coated (cuvettes and probes) were either (i) filled with 600 μl of the coating composition and excess coating composition was removed after about 10 s (cuvettes), or (ii) dipped into the coating composition for about 2 s (probes). Articles (cuvettes and probes) were subsequently dried in air for about 1 hour.

(20) In general, improvements of blood adhesion to the polymeric surfaces of articles used for thrombelastographic diagnostics by applying coatings according to the present invention can be detected by comparing the initially achieved maximum clot firmness with the reduction of clot firmness at the end of the measurement (e.g., 60 min after measurement start). This ratio, also called “ML” parameter (maximum lysis activity, ML; % ratio between (i) clot firmness at the end of measurement, for example 60 minutes after measurement start, and (ii) maximum clot firmness), can be artificially lowered by partial ruptures of the fibrin network from the surface during measurements (see FIG. 7). Since the ML parameter is often used for the diagnosis of hyperfibrinolytic activity in the coagulation system of a patient blood sample, lower values due to insufficient surface adhesion of the fibrin network are a potential risk in haemostasis analysis. Since the blood of patients with considerably increased platelet content tends to tear off the surface due to denser clot packing, mistakable measurements cannot be excluded. By applying coatings for the articles according to the present invention, this drawback of unwanted tear-offs can be satisfactorily eliminated (see also the following examples and FIG. 8-10). It can also appear that the tear-off of a blood clot starts even before the maximum clot firmness is achieved in the measurement (maximum clot firmness is typically achieved about 20-30 minutes after initial clotting). In this case, the ML parameter might be less influenced by the tear-off, but the clot firmness amplitude measured 20 minutes after initial clotting (called parameter A20) will be reduced. Accordingly, occurrence of an unwanted tear-off of the blood clot from the article surface can either be detected by a higher ML parameter and/or a lower A20 parameter when comparing to an article with improved surface adhesion of the blood clot. Therefore, higher ML values (as compared to a reference) and/or lower A20 values (as compared to a reference) indicate increased adhesion to clotting blood (as compared to the reference).

(21) The functionality was assessed by comparing thrombelastographic measurements performed with ROTEG® 05 devices (Pentapharm GmbH, Germany), where differently treated articles with dimensions comparable to the corresponding original measurement articles (ROTEM® Cup&Pin Pro, Tem International GmbH, Germany) were compared regarding clot firmness amplitudes after 20 minutes (A20) and maximum lysis activity (ML; % ratio between clot firmness 60 minutes after measurement start and maximum clot firmness).

(22) Measurements were performed by pipetting 20 μl of extrinsic activator (ex-TEM®, Tem International GmbH, Germany) and 20 μl of 200 mM CaCl.sub.2 (star-TEM®, Tem International GmbH, Germany) to a 300 μl citrated blood sample and transferring it to the respective article.

Example 2: Comparison of Uncoated Articles

(23) In order to efficiently determine and compare the surface characteristics regarding blood adhesion of various polymer materials, untreated/uncoated articles (cups and pins) made of polymethylpentene (PMP; TPX®, Mitsui & Co. Ltd., Japan), methyl methacrylate acrylonitrile butadiene styrene (MABS; Terlux® 2802, INEOS Styrolution Group GmbH, Germany), polyamide (PA; Trogamid® T5000, Evonik Industries AG, Germany), polyphenylene sulfide (PPS; Ryton® R-4, SOLVAY GmbH, Germany), or polyurethane (PU; Desmopan® 385 S, Bayer MaterialScience AG, Germany) were obtained by industrial injection molding. Those untreated/uncoated articles underwent functionality testing as described above (cf. Example 1). Results are shown in FIGS. 7 (clot firmness amplitudes after 20 minutes; A20) and 8 (maximum lysis activity; ML).

(24) Injection-molded articles made of MABS, PA or PPS show significantly higher A20 values and significantly lower ML values as compared to injection-molded articles made of PMP or PU (FIG. 7, 8). Those results indicate that MABS, PA or PPS represent suitable coating polymers, which can improve surface adhesion to clotting blood, in particular if a suitable nonpolar solvent is used. Examples of such a suitable non-polar solvents are the “lower risk” solvents n-propanol (in particular for PA), xylene (in particular for MABS) and/or ethylbutanol for (in particular for MABS).

(25) Articles made of PMP or PU without any treatment or coating show poor results regarding blood clot adhesion as indicated by comparably low A20 values and comparably high ML mean values in thromboelastometric measurements (FIG. 7, 8).

Example 3: Coating of an Exemplary Article Made of Polymethylpentene (PMP)

(26) Uncoated articles (cup and pin) made of polymethylpentene (PMP; TPX®, Mitsui & Co. Ltd., Japan) was obtained by industrial injection molding. The uncoated articles were then partially coated with MABS (in ethylbutanol).

(27) Thereafter, the article coated with MABS as well as an uncoated article (made of polymethylpentene (PMP; TPX®, Mitsui & Co. Ltd., Japan)) underwent functionality testing as described above (Example 1), whereby instead of A20 and ML parameters, typical thromboelastography traces were obtained as shown in FIG. 9.

(28) The thromboelastography trace of the untreated/uncoated article (cuvette and probe) made of Polymethylpentene (PMP; TPX®, Mitsui & Co. Ltd., Japan) is shown in FIG. 9A. This thromboelastography trace of the untreated/uncoated article reflects partial “tear-offs” (FIG. 9A), whereas the thromboelastography trace obtained with an article made of identical material (PMP; TPX®, Mitsui & Co. Ltd., Japan), but coated with a coating composition according to the present invention, which comprises MABS (Terlux®, INEOS Styrolution Group GmbH, Germany), shown in FIG. 9B shows no such “tear-offs” (FIG. 9B).

(29) In summary, the uncoated (“untreated”) articles made of PMP shows undesired “tear-offs” (FIG. 9A), whereas those undesired “tear-offs” were abolished if the article was coated with MABS (FIG. 9B). This result demonstrates the improved surface functionality regarding clot adhesion as provided by the polymer coated onto the surface of the article.

Example 4: Coating of Further Articles Made of Polymethylpentene (PMP)

(30) To determine blood clot adhesion of different coatings, uncoated articles (cups and pins) made of polymethylpentene (PMP; TPX®, Mitsui & Co. Ltd., Japan) were obtained by industrial injection molding and were partially coated with MABS (in xylene), HIPS, ABS, PMMA, or PC as described above (cf. Example 1).

(31) Alternatively, uncoated articles were treated with a xylol/ethylbutanol solvent mixture (50 vol.-% xylol; 50 vol.-% ethylbutanol) without any polymer dissolved. Those articles served as comparative example to evaluate the effects of the “coating” with pure solvent (i.e., without any polymer contained therein).

(32) Thereafter, PMP articles coated with MABS, HIPS, ABS, PMMA or PC; PMP articles treated with xylol/ethylbutanol; and uncoated PMP articles underwent functionality testing as described above (cf. Example 1). Results are shown in FIGS. 10 (clot firmness amplitudes after 20 minutes; A20) and 11 (maximum lysis activity; ML).

(33) In summary, uncoated (“untreated”) articles made of PMP show lower mean values for A20 and higher mean values for ML as compared to PMP articles coated with MABS, HIPS, ABS, PMMA, or PC. Accordingly, surface coating with MABS, HIPS, ABS, PMMA or PC resulted in considerable improvements regarding A20 and ML parameters as compared to uncoated PMP articles (FIG. 10, 11). The most pronounced and significant improvements were obtained with a coating comprising MABS, HIPS, ABS or PC.

(34) Surface treatment of PMP articles with compositions comprising the solvent only (but no polymer) resulted in A20 and ML values comparable to those of untreated/uncoated articles (FIG. 10, 11). This result demonstrates that the improved surface functionality regarding clot adhesion is indeed provided by the polymer coated onto the surface of the articles and largely independent from the solvent used.

Example 5: Coating of Articles Made of Methyl Methacrylate Acrylonitrile Butadiene Styrene (MABS)

(35) To determine whether articles made of materials, which already show good blood clot adhesion, can be further improved by applying a coating, uncoated articles (cups and pins) made of methyl methacrylate acrylonitrile butadiene styrene (MABS; Terlux® 2802, INEOS Styrolution Group GmbH, Germany) were obtained by industrial injection molding and were coated with MABS (in xylene) or ABS as described above.

(36) Thereafter, MABS articles coated with MABS (in xylene) or ABS as well as uncoated articles underwent functionality testing as described above. Results are shown in FIGS. 12 (clot firmness amplitudes after 20 minutes; A20) and 13 (maximum lysis activity; ML).

(37) In summary, uncoated (“untreated”) articles made of MABS show lower mean values for A20 and higher mean values for ML as compared to MABS articles coated with MABS or ABS. Accordingly, surface coating with MABS or ABS resulted in improvements regarding A20 and ML when compared to uncoated MABS articles (FIG. 12, 13). However, due to the already quite good surface properties of MABS itself, the effects of the coatings are less pronounced as compared to articles made of PMP (cf. example 4).

(38) Surprisingly, coating of articles made of MABS (Terlux® 2802, INEOS Styrolution Group GmbH, Germany) with exactly the same MABS material dissolved in xylene (and/or ethylbutanol) also resulted in significant improvements regarding A20 and ML (see FIG. 12, 13). These results imply that the surface properties regarding blood adhesion are not only improved by using a coating material providing better surface properties than the material of the uncoated article, but also by the process of coating. Namely, coating with a dissolved polymer provides improved surface characteristics as compared to injection molding of the same material. Without being limited thereto, the inventors assume that a potential reason may be a different alignment of molecules on the surface (e.g., randomly vs. ordered), or the appearance of impurities on the surface of injection-molded surfaces due to lubricant residuals of the molding machinery.