Low-volume coagulation assay
Compositions and methods for measuring coagulation parameters using very small volumes of blood are provided. Advantageously, the methods described herein can be performed from a single drop of blood (about 20 μL) while generally leaving enough sample to perform other measurements, optionally in a multiplexed format. The methods and devices do not require a skilled operator and can be performed at the point of service, which can be an important feature for managing blood coagulation disorders and treatments thereof.
1. A method for measuring a plurality of coagulation parameters of a blood sample of a subject, the method comprising:
(a) obtaining a blood sample having a volume of less than or about 1 ml;
(b) inserting a cartridge containing said blood sample and containing all reagents required for performing a plurality of automated coagulation assays into a device for performing said plurality of automated coagulation assays, said device comprising automated sample processing mechanisms including an automated fluid transfer apparatus, wherein said automated fluid transfer apparatus is configured to move tips and is configured to move a volume of sample within the device, wherein said tips comprise a collection tip and an assay unit tip;
(c) distributing at least a portion of the sample in said collection tip;
(d) performing the plurality of automated coagulation assays with said sample to measure said plurality of coagulation parameters in the blood sample or a portion or portions thereof, wherein said performing comprises multiplexed performance of the automated coagulation assays in parallel, and wherein the plurality of automated coagulation assays comprises:
i) a coagulation time assay, and
ii) at least one automated coagulation assay for a coagulation parameter other than coagulation time;
wherein said coagulation assay comprises:
(e) using said automated fluid transfer apparatus, combining a plurality of beads or other particles with said blood sample in said assay unit tip to provide a sample mixture and moving said sample mixture in the assay unit tip near to an optical detector;
(f) obtaining, subsequent to step (e), a set of images comprising individual images, said set of images comprising images of said beads or other particles and sample, or a portion or portions thereof, obtained during performance of said coagulation time assay;
(g) determining bead or other particle mobility at different times, where said determining comprises determining the rate of settling of said beads or other particles with said set of images; and
(h) analyzing the set of images to measure the coagulation time of the blood sample, or of a portion or portions thereof, wherein said analyzing comprises locating the time point when a transition of the mobility of the beads or other particles within the blood sample occurs.
2. The method of claim 1 , wherein said at least one automated coagulation assay for a coagulation parameter other than coagulation time is an assay selected from the group of automated coagulation assays consisting of: Activated Partial Thromboplastin Time (aPTT) assay, prothrombin time (PT) assay, International Normalized Ratio (INR) assay, amount of fibrinogen assay, bleeding time assay, coagulation factor concentration assay, anti-phospholipid antibody detection assay, dilute Russell's viper venom time (dRVVT) assay, platelet function assay, platelet count assay, and euglobulin lysis time (ELT) assay.
3. The method of claim 1 , wherein less than about 2 μl of the sample is utilized for an individual assay of said plurality of assays.
4. The method of claim 1 , wherein the reaction volume of each of said plurality of assays is about or less than 60 μl.
5. The method of claim 1 , wherein the amount of time for carrying out steps (a) to (b) is less than or about 1 hour.
6. The method of claim 1 , wherein an individual image of said set of images is pixilated and comprises at least 10,000 pixels.
7. The method of claim 1 , further comprising diluting said blood sample such that the coagulation time of the blood sample after dilution is between about 1 minute and about 10 minutes.
8. The method of claim 7 , further comprising adding fibrinogen to the sample, or portion or portions thereof.
9. The method of claim 1 , wherein said blood sample is obtained via a non-venous route.
10. The method of claim 1 , wherein said images are light scattering images of the coagulation reaction.
11. The method of claim 1 , wherein the coagulation time is measured based on a transition of the intensity of scattered light.
12. The method of claim 1 , wherein said plurality of beads or other particles comprise beads or other particles of at least two different sizes.
13. The method of claim 1 , wherein said step of analyzing said set of images comprises locating a time point when said beads or other particles become substantially motionless.
14. The method of claim 1 , wherein said beads or other particles are labeled.
15. The method of claim 14 , wherein said beads or other particles are labeled with a fluorescent label.
16. The method of claim 1 , wherein less than about 10 μl of the sample is utilized for an individual assay of said plurality of assays.