Activation assay for the diagnosis of a heparin-induced thrombocytopenia

Abstract

The present invention relates to a functional, easily automatable assay for establishing a heparin-induced thrombocytopenia (HIT). What is measured is the secretion of PF4 (platelet factor 4) from activated thrombocytes.

Claims

1. A method for detecting anti-platelet factor 4(PF4)/heparin complex antibodies in a body-fluid sample, the method comprising: i. providing a reaction mixture by mixing the sample with a thrombocyte-containing reagent and with a PF4-binding, unbranched polysaccharide or with a PF4-binding polyanion; ii. incubating the reaction mixture; iii. determining an amount of PF4 in the reaction mixture; iv. comparing the amount of PF4 in the reaction mixture with a predetermined reference value for the amount of PF4 in reaction mixtures containing body-fluid samples from donors known to contain no anti-PF4/heparin complex antibodies; and v. establishing the presence of anti-PF4/heparin complex antibodies in the sample when the amount of PF4 in the reaction mixture exceeds the reference value.

2. The method of claim 1, wherein the thrombocyte-containing reagent contains human thrombocytes from one or more body-fluid samples from healthy donors known to contain no anti-PF4/heparin complex antibodies.

3. The method of claim 1, wherein the amount of PF4 in the reaction mixture is determined by contacting at least one anti-PF4 antibody with the reaction mixture.

4. The method of claim 1, wherein a first and a second anti-PF4 antibody and a first and a second component of a signal-forming system, which interact such that a detectable signal is formed when the first and the second component of the signal-forming system are brought into close proximity to one another, are mixed with the reaction mixture and wherein the first anti-PF4 antibody is in an associated state with the first component of the signal-forming system or is associated therewith during the incubation of the reaction mixture and wherein the second anti-PF4 antibody is in an associated state with the second component of the signal-forming system or is associated therewith during the incubation of the reaction mixture.

5. The method of claim 4, wherein the first and second component of the signal-forming system comprise in each case a particulate solid phase, and wherein the agglutination of the particulate solid phases in the reaction mixture is measured.

6. The method of claim 4, wherein the first component of the signal-forming system is a chemiluminescent agent and the second component of the signal-forming system is a photosensitizer or vice versa and wherein the chemiluminescence in the reaction mixture is measured.

7. The method of claim 4, in which the provided reaction mixture is not subjected to a centrifugation step for the removal of thrombocytes.

8. The method of claim 1, wherein the presence of anti-PF4/heparin complex antibodies in the sample is established when the amount of PF4 in the reaction mixture in step iv exceeds the reference value by about 2-fold or greater.

9. The method of claim 1, wherein the presence of anti-PF4/heparin complex antibodies in the sample is established when the amount of PF4 in the reaction mixture in step iv exceeds the reference value by between about 2-fold and about 10-fold.

10. The method of claim 1, wherein the thrombocyte-containing reagent comprises thrombocytes from healthy donors.

Description

EXAMPLES

Example 1

Homogeneous Immunoassay for the Detection of Anti-PF4/Heparin Complex Antibodies

(1) The LOCI® technology used here involves bringing a latex particle-coupled chemiluminescent compound (Chemibeads) and a latex particle-coupled photosensitizer (Sensibeads) into close proximity to one another through simultaneous binding to an analyte, with the result that singlet oxygen, which is produced by the photosensitizer, can stimulate the chemiluminescent compound.

(2) 2 μL of heat-inactivated (at 56° C., 30 min) human plasma sample were mixed with 7.5 μL of a suspension containing washed human thrombocytes from 6 healthy donors (approximately 300×10.sup.9 thrombocytes/L) and 10 μL of a buffer solution containing 0.2 IU/mL heparin and incubated at 37° C. After 10 minutes, the following components were added to the reaction mixture: 50 μL of a “Chemibead” solution containing, in a buffer solution, latex particles coated with a chemiluminescent compound (2-(4-(N,N,ditetradecyl)-anilino-3-phenyl thioxene) and a first monoclonal anti-PF4 antibody (MAK-23/064) which binds both free and complexed PF4 protein (50 μg/mL); 50 μL of an antibody solution containing a second biotinylated monoclonal anti-PF4 antibody (MAK-23/074) which binds both free and complexed PF4 protein (5 μg/mL); and 100 μL of a “Sensibead” solution containing, in a buffer solution, latex particles coated with a photosensitizer compound (bis-(trihexyl)-silicon-t-butyl-phthalocyanine) and streptavidin (50 μg/mL).

(3) After about 10 minutes, the chemiluminescent signal was measured [kcounts].

(4) The method according to the invention (also called “PF4 release” hereinafter) was used to measure plasma samples from 10 HIT patients, for whom a HIT had been diagnosed on the basis of clinical criteria (4 T score, in some cases with thrombotic event) and the existence of anti-PF4/heparin complex antibodies had been established using two independent, commercially available immunoassays (HemosIL® AcuStar HIT-Ab(PF4-H), Instrumentation Laboratories, and Asserachrom® HPIA-IgG, Diagnostica Stago).

(5) Furthermore, the method according to the invention was used to measure plasma samples from 7 healthy donors (who do not have clinical HIT criteria and also do not have anti-PF4/heparin complex antibodies) and to measure a normal plasma pool (from about 20 plasmas from healthy donors, “FNP”).

(6) As “100% control” for the PF4 release assay, an antibody solution containing a thrombocyte-activating anti-PF4/heparin complex antibody (50 μg/mL) was used instead of a plasma sample in order to ascertain the maximum possible secretion of PF4 with the thrombocyte-containing reagent used. To ascertain the secretion of PF4 in % (PF4 release [%]), the raw values measured in kcounts were converted to a proportion of the raw value of the 100% control.

(7) For the purposes of comparison, the stated samples were also measured using the [14C]-serotonin release assay (also called “SRA” hereinafter), in accordance with Sheridan, D. et al. (1986) A diagnostic test for heparin-induced thrombocytopenia. Blood 67 (1): 27-30, which is considered the gold standard.

(8) The assay results are compiled in Table 1.

(9) TABLE-US-00001 TABLE 1 Comparison of the PF4 release assay according to the invention with the SRA assay for HIT diagnosis PF4 release PF4 release SRA Thrombot. Sample ID [kcounts] [%] [%] event HIT HIT-50 337.1 15 <20 No patients HIT-38 534.4 24 38 No HIT-33 757.9 34 55 Yes HIT-6 739.8 33 64 Yes HIT-17 867.3 39 69 Yes HIT-20 1295.4 58 85 Yes HIT-12 1373.7 62 88 Yes HIT-34 1387.8 62 93 Yes HIT-2 1814.4 81 96 Yes HIT-28 2201.4 99 100 Yes 100% control 2232.4 100 N/A N/A Healthy 8864 182.6 8 <10 No donors 8098 229.0 10 <10 No 8848 158.0 7 <10 No 8408 177.5 8 <10 No 8396 169.7 8 <10 No 8866 174.5 8 <10 No 8784 210.8 9 <10 No FNP 161.7 7 <10 No Buffer blank 162.2 7 N/A N/A

(10) It becomes apparent that the results of the method according to the invention correlate very well with the results of the SRA gold-standard assay. In all the reaction mixtures containing HIT patient samples, it is possible to detect a PF4 concentration which is significantly increased compared to healthy donors. As threshold (cut-off) for the differentiation of samples containing anti-PF4/heparin complex antibodies from those containing none, it would be possible to define a PF4 release value of ≥15% or even ≥20% (cut-off of SRA assay: ≥20%). However, for a statistically more precise definition of the cut-off value, a distinctly higher number of sample measurements is required.

EMBODIMENTS

Embodiment 1

(11) A method for detecting anti-PF4/heparin complex antibodies in a body-fluid sample, the method comprising the steps: i. providing a reaction mixture by mixing the sample with a thrombocyte-containing reagent and with a PF4-binding, unbranched polysaccharide or with a PF4-binding polyanion; ii. incubating the reaction mixture; and then iii. determining the amount of PF4 in the reaction mixture; iv. comparing the thus determined amount of PF4 in the reaction mixture with a predetermined reference value for the amount of PF4 in reaction mixtures containing body-fluid samples from donors known to contain no anti-PF4/heparin complex antibodies; and v. establishing the presence of anti-PF4/heparin complex antibodies in the sample when the amount of PF4 that is determined in the reaction mixture exceeds the reference value.

Embodiment 2

(12) The method as in Embodiment 1, wherein the thrombocyte-containing reagent contains human thrombocytes from one or more healthy donors known to contain no anti-PF4/heparin complex antibodies.

Embodiment 3

(13) The method as in either of Embodiments 1 and 2, wherein the amount of PF4 in the reaction mixture is determined by contacting at least one anti-PF4 antibody with the reaction mixture.

Embodiment 4

(14) The method as in Embodiment 3, wherein a first and a second anti-PF4 antibody and a first and a second component of a signal-forming system, which interact such that a detectable signal is formed when the first and the second component of the signal-forming system are brought into close proximity to one another, are mixed with the reaction mixture and wherein the first anti-PF4 antibody is in an associated state with the first component of the signal-forming system or is associated therewith during the incubation of the reaction mixture and wherein the second anti-PF4 antibody is in an associated state with the second component of the signal-forming system or is associated therewith during the incubation of the reaction mixture.

Embodiment 5

(15) The method as in Embodiment 4, wherein the first and second component of the signal-forming system comprise in each case a particulate solid phase, and wherein the agglutination of the particulate solid phases in the reaction mixture is measured.

Embodiment 6

(16) The method as in Embodiment 4, wherein the first component of the signal-forming system is a chemiluminescent agent and the second component of the signal-forming system is a photosensitizer or vice versa and wherein the chemiluminescence in the reaction mixture is measured.

Embodiment 7

(17) The method as in any of Embodiments 4 to 6, in which the provided reaction mixture is not subjected to a centrifugation step for the removal of the thrombocytes.

Embodiment 8

(18) A method for diagnosing a heparin-induced thrombocytopenia, wherein a method as in any of Embodiments 1 to 7 is used to detect the presence of anti-PF4/heparin complex antibodies in a body-fluid sample from a patient.

Embodiment 9

(19) An assay kit for carrying out a method as in any of Embodiments 1 to 8, containing the following components: a. a first reagent containing thrombocytes; b. a second reagent containing a PF4-binding, unbranched polysaccharide or a PF4-binding polyanion; and c. one or more reagents for the detection of PF4, with at least one reagent containing an anti-PF4 antibody.

Embodiment 10

(20) The assay kit as in Embodiment 9, wherein the second reagent contains a PF4-binding, unbranched polysaccharide from the group consisting of heparin, unfractionated heparin, fractionated heparin, dextran sulfate and fucoidan; or a PF4-binding polyanion from the group consisting of polyvinyl sulfate, polyvinyl sulfonate, polyvinyl phosphate, polyvinyl phosphonate, polystyrene sulfate and polystyrene sulfonate.