Methods and kits for detecting heparin/platelet factor 4 antibodies

1 . A method of detecting heparin/platelet factor 4 antibodies to diagnose heparin-induced thrombocytopenia (HIT) in a subject suspected of having HIT, said method comprising: 
 (a) contacting a sample obtained from the subject with particles to form a test mixture, said particles being complexed to platelet factor 4 (PF4), which particle-complexed PF4 reacts specifically with heparin/platelet factor 4 antibodies, and said particles forming specific aggregates upon contacting heparin/platelet factor 4 antibodies; and    (b) analyzing the test mixture to detect said specific aggregates using a particulate immunofiltration assay,    wherein the presence of said specific aggregates indicates HIT in the subject.    
   
   
       2 . The method of  claim 1 , wherein the particulate immunofiltration assay comprises: 
 (a) passing the test mixture through a filter means comprising apertures which are larger than the particles but smaller than the specific aggregates, thereby producing a filtrate; and    (b) passing said filtrate through a wicking means adjacent and in fluid communication with the filter means, said wicking means separating any non-specifically aggregated particles and/or any unaggregated particles from said specific aggregates, wherein the unaggregated particles migrate horizontally through said wicking means at a rate faster than said specific aggregates,    wherein the presence of non-specifically aggregated or unaggregated particles in the filtrate indicates the absence of heparin/platelet factor 4 antibodies in the sample and the absence of non-specifically aggregated or unaggregated latex particles in the filtrate indicates the presence of the heparin/platelet factor 4 antibodies in the sample.    
   
   
       3 . A method for detecting the presence of heparin/platelet factor 4 antibodies in a sample, said method comprising: 
 (a) forming a test mixture by incubating the sample with particles complexed to platelet factor 4 (PF4), which particle-complexed PF4 reacts specifically with heparin/platelet factor 4 antibodies, and said particles forming specific aggregates upon contacting heparin/platelet factor 4 antibodies;    (b) passing the test mixture through a filter means comprising apertures which are larger than the particles but smaller than the specific aggregates, thereby producing a filtrate; and    (c) passing said filtrate through a wicking means adjacent and in fluid communication with the filter means, said wicking means separating any non-specifically aggregated particles and/or any unaggregated particles from the specific aggregates, wherein the unaggregated particles and non-specifically aggregated particles migrate horizontally through said wicking means at a rate faster than said specific aggregates,    wherein the presence of non-specifically aggregated or unaggregated particles in the filtrate indicates the absence of heparin/platelet factor 4 antibodies in the sample and the absence of non-specifically aggregated or unaggregated particles in the filtrate indicates the presence of heparin/platelet factor 4 antibodies in the sample.    
   
   
       4 . The method of  claim 2  or  3 , wherein the sample comprises a mammalian bodily fluid.  
   
   
       5 . The method of  claim 2  or  3 , wherein the sample comprises a human bodily fluid.  
   
   
       6 . The method of  claim 2  or  3 , wherein the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.  
   
   
       7 . The method of  claim 2  or  3 , wherein the particles have a visually recognizable color which can be ascertained by the unaided eye.  
   
   
       8 . The method of  claim 2  or  3 , wherein the particles have a mean diameter from about 0.1 micrometers to about 10 micrometers.  
   
   
       9 . The method of  claim 8 , wherein the particles have a mean diameter from about 0.2 micrometers to about 0.6 micrometers.  
   
   
       10 . The method of  claim 2  or  3 , wherein the particles are microspheres.  
   
   
       11 . The method of  claim 2  or  3 , wherein the particles are non-spherical.  
   
   
       12 . The method of  claim 2  or  3 , wherein the particles are anionic.  
   
   
       13 . The method of  claim 2  or  3 , wherein the particles comprise polystyrene.  
   
   
       14 . The method of  claim 2  or  3 , wherein the particles comprise latex.  
   
   
       15 . The method of  claim 14 , wherein the particles comprise latex in the concentration of from about 0.01% w/v to about 2% w/v.  
   
   
       16 . The method of  claim 15 , wherein the particles comprise latex in the concentration of from about 0.3% w/v to about 0.4% w/v.  
   
   
       17 . The method of  claim 2  or  3 , wherein the particles comprise metal colloid.  
   
   
       18 . The method of  claim 17 , wherein the metal colloid is gold colloid.  
   
   
       19 . The method of  claim 2  or  3 , wherein the particles have been dried.  
   
   
       20 . The method of  claim 2  or  3 , wherein the particles are sealed in a glass ampoule.  
   
   
       21 . The method of  claim 2  or  3 , wherein particles are stabilized with a colloidal stabilizer.  
   
   
       22 . The method of  claim 21 , wherein the colloidal stabilizer comprises sodium tripolyphosphate.  
   
   
       23 . The method of  claim 22 , wherein the sodium tripolyphosphate is in the concentration range of from about 0.001% w/v to about 0.1% w/v.  
   
   
       24 . The method of  claim 23 , wherein the sodium tripolyphosphate is in the concentration range of from about 0.01% w/v to about 0.1% w/v.  
   
   
       25 . The method of  claim 21 , wherein the colloidal stabilizer comprises an anionic detergent.  
   
   
       26 . The method of  claim 25 , wherein the anionic detergent comprises sodium dodecyl sulphate.  
   
   
       27 . The method of  claim 25 , wherein the anionic detergent comprises sodium laurel sarcosine.  
   
   
       28 . The method of  claim 25 , wherein the anionic detergent comprises polyoxyethylene sorbitan monolaureate.  
   
   
       29 . The method of  claim 28 , wherein the polyoxyethylene sorbitan monolaureate is in the concentration range of from about 0.0001% w/v to about 0.1% w/v.  
   
   
       30 . The method of  claim 29 , wherein the polyoxyethylene sorbitan monolaureate is in the concentration range of from about 0.001% w/v to about 0.01% w/v.  
   
   
       31 . The method of  claim 21 , wherein the colloidal stabilizer comprises sodium polymetaphosphate, sodium phosphate glass, sodium pyrophosphate or other polyphosphate molecules.  
   
   
       32 . The method of clam  21 , wherein the colloidal stabilizer comprises nonionic detergents.  
   
   
       33 . The method of  claim 21 , wherein the colloidal stabilizer comprises a polyphosphate and one or more detergents.  
   
   
       34 . The method of  claim 2  or  3 , wherein a reaction enhancer solution is further added to the test mixture.  
   
   
       35 . The method of  claim 34 , wherein the reaction enhancer solution has a pH of 7.2.  
   
   
       36 . The method of  claim 34 , wherein the reaction enhancer solution comprises polyethylene glycol, sodium chloride, and glycine.  
   
   
       37 . The method of  claim 36 , wherein the polyethylene glycol is polyethylene glycol 8000.  
   
   
       38 . The method of  claim 37 , wherein the polyethylene glycol 8000 is in the range of from about 5% w/v to about 15% w/v.  
   
   
       39 . The method of  claim 38 , wherein the polyethylene glycol 8000 is in the range of from about 8% w/v to about 12% w/v.  
   
   
       40 . The method of  claim 36 , wherein the sodium chloride is in the range of from about 0.0% w/v to about 1% w/v.  
   
   
       41 . The method of  claim 40 , wherein the sodium chloride is in the range of from about 0.0% w/v to about 0.1% w/v.  
   
   
       42 . The method of  claim 36 , wherein they glycine is in the range of from about 0.01 molar to about 0.2 molar.  
   
   
       43 . The method of  claim 42 , wherein they glycine is in the range of from about 0.02 molar to about 0.1 molar.  
   
   
       44 . The method of  claim 2  or  3 , wherein the filter means comprises a controlled pore polycarbonate membrane.  
   
   
       45 . The method of  claim 44 , wherein the controlled pore polycarbonate membrane has a pore size from about 2 micrometers to about 12 micrometers.  
   
   
       46 . The method of  claim 45 , wherein the controlled pore polycarbonate membrane has a pore size of about 3 micrometers.  
   
   
       47 . The method of  claim 2  or  3 , wherein the wicking means comprises non-woven fibers of glass or synthetic polymeric material  
   
   
       48 . The method of  claim 2  or  3 , wherein the presence of the heparin/platelet factor 4 antibodies is visually determined by the color of the filtrate.  
   
   
       49 . The method of  claim 2  or  3 , wherein the particulate immunofiltration assay is performed in a reaction cell.  
   
   
       50 . A kit for detecting heparin/platelet factor 4 antibodies in a liquid sample from a subject to diagnose heparin-induced thrombocytopenia (HIT) in said subject suspected of having HIT, said kit comprising: 
 (a) a reaction cell comprising particles complexed to platelet factor 4 (PF4), which particle-complexed PF4 reacts specifically with heparin/platelet factor 4 antibodies, and said particles forming specific aggregates upon contacting heparin/platelet factor 4 antibodies; and    (b) an assay plate comprising: 
 (i) a top member having a filter well and an observation well that is at a fixed distance from said filter well, said filter well being adapted to receive the liquid sample;  
 (ii) a filter means adjacent the top member and extending across the filter well comprising a controlled pore membrane for effecting a first separation of said specific aggregates from any non-specifically aggregated particles and/or any unaggregated particles, wherein said non-specifically aggregated and/or said unaggregated particles migrate vertically through the filter means;  
 (iii) a wicking means adjacent and in fluid communication with the filter means and extending the length of the filter well and the observation well, said wicking means consisting essentially of a plurality of fibers for effecting a second separation of said specific aggregates which passed through said filter means from said non-specifically aggregated particles and/or said unaggregated particles, wherein any unaggregated particles migrate horizontally through said wicking means at a rate faster than said specific aggregates do, and said unaggregated particles are directly visually detectable through said observation well; and  
   (iv) a bottom member adjacent the wicking means; wherein said assay plate is free of non-mobilizable ligand-specific binder,    wherein the detection of the absence of unaggregated particles in said observation well indicates the presence of heparin/platelet factor 4 antibodies in said subject thereby diagnosing HIT in the subject.    
   
   
       51 . The kit of  claim 50 , wherein the sample comprises a mammalian bodily fluid.  
   
   
       52 . The kit of  claim 50 , wherein the sample comprises a human bodily fluid.  
   
   
       53 . The kit of  claim 50 , wherein the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.  
   
   
       54 . The kit of  claim 50 , wherein the particles have a visually recognizable color which can be ascertained by the unaided eye.  
   
   
       55 . The kit of  claim 50 , wherein the particles have a mean diameter from about 0.1 micrometers to about 1.0 micrometers.  
   
   
       56 . The kit of  claim 55 , wherein the particles have a mean diameter from about 0.2 micrometers to about 0.6 micrometers.  
   
   
       57 . The kit of  claim 50 , wherein the particles are microspheres.  
   
   
       58 . The kit of  claim 50 , wherein the particles are non-spherical.  
   
   
       59 . The kit of  claim 50 , wherein the particles are anionic.  
   
   
       60 . The kit of  claim 50 , wherein the particles comprise polystyrene.  
   
   
       61 . The kit of  claim 50 , wherein the particles comprise latex.  
   
   
       62 . The kit of  claim 61 , wherein the particles comprise latex in the concentration of from about 0.01% w/v to about 2% w/v.  
   
   
       63 . The kit of  claim 62 , wherein the particles comprise latex in the concentration of from about 0.3% w/v to about 0.4% w/v.  
   
   
       64 . The kit of  claim 50 , wherein the particles comprise metal colloid.  
   
   
       65 . The kit of  claim 64 , wherein the metal colloid is gold colloid.  
   
   
       66 . The kit of  claim 50 , wherein the particles have been dried.  
   
   
       67 . The kit of  claim 50 , wherein the particles are sealed in a glass ampoule.  
   
   
       68 . The kit of  claim 50 , wherein particles are stabilized with a colloidal stabilizer.  
   
   
       69 . The kit of  claim 68 , wherein the colloidal stabilizer comprises sodium tripolyphosphate.  
   
   
       70 . The kit of  claim 69 , wherein the sodium tripolyphosphate is in the concentration range of from about 0.001% w/v to about 0.1% w/v.  
   
   
       71 . The kit of  claim 70 , wherein the sodium tripolyphosphate is in the concentration range of from about 0.01% w/v to about 0.1% w/v.  
   
   
       72 . The kit of  claim 68 , wherein the colloidal stabilizer comprises an anionic detergent.  
   
   
       73 . The kit of  claim 72 , wherein the anionic detergent comprises sodium dodecyl sulphate.  
   
   
       74 . The kit of  claim 72 , wherein the anionic detergent comprises sodium laurel sarcosine.  
   
   
       75 . The kit of  claim 72 , wherein the anionic detergent comprises polyoxyethylene sorbitan monolaureate.  
   
   
       76 . The kit of  claim 75 , wherein the polyoxyethylene sorbitan monolaureate is in the concentration range of from about 0.0001% w/v to about 0.1% w/v.  
   
   
       77 . The kit of  claim 76 , wherein the polyoxyethylene sorbitan monolaureate is in the concentration range of from about 0.0.001% w/v to about 0.01% w/v.  
   
   
       78 . The kit of  claim 68 , wherein the colloidal stabilizer comprises sodium polymetaphosphate, sodium phosphate glass, sodium pyrophosphate or other polyphosphate molecules.  
   
   
       79 . The kit of clam  68 , wherein the colloidal stabilizer comprises nonionic detergents.  
   
   
       80 . The kit of  claim 68 , wherein the colloidal stabilizer comprises a polyphosphate and one or more detergents.  
   
   
       81 . The kit of  claim 50 , wherein the reaction cell further comprises a reaction enhancer solution is further added to the test mixture.  
   
   
       82 . The kit of  claim 81 , wherein the reaction enhancer solution has a pH of 7.2.  
   
   
       83 . The kit of  claim 81 , wherein the reaction enhancer solution comprises polyethylene glycol, sodium chloride, and glycine.  
   
   
       84 . The kit of  claim 83 , wherein the polyethylene glycol is polyethylene glycol 8000.  
   
   
       85 . The kit of  claim 84 , wherein the polyethylene glycol 8000 is in the range of from about 5% w/v to about 15% w/v.  
   
   
       86 . The kit of  claim 85 , wherein the polyethylene glycol 8000 is in the range of from about 8% w/v to about 12% w/v.  
   
   
       87 . The kit of  claim 83 , wherein the sodium chloride is in the range of from about 0.0% w/v to about 1% w/v.  
   
   
       88 . The kit of  claim 87 , wherein the sodium chloride is in the range of from about 0.0% w/v to about 0.1% w/v.  
   
   
       89 . The kit of  claim 83 , wherein they glycine is in the range of from about 0.01 molar to about 0.2 molar.  
   
   
       90 . The kit of  claim 89 , wherein they glycine is in the range of from about 0.02 molar to about 0.1 molar.  
   
   
       91 . The kit of  claim 50 , wherein the filter membrane comprises a controlled pore polycarbonate membrane.  
   
   
       92 . The kit of  claim 91 , wherein the controlled pore polycarbonate membrane has a pore size from about 2 micrometers to about 12 micrometers.  
   
   
       93 . The kit of  claim 92 , wherein the controlled pore polycarbonate membrane has a pore size of about 3 micrometers.  
   
   
       94 . The kit of  claim 50 , wherein the wicking means comprises non-woven fibers of glass or synthetic polymeric material  
   
   
       95 . The method of  claim 2  or  3 , wherein the particulate immunofiltration assay is performed in a reaction cell.  
   
   
       96 . A kit for detecting heparin/platelet factor 4 antibodies in a sample to diagnose heparin-induced thrombocytopenia (HIT) in a subject suspected of having HIT, said kit comprising: 
 (a) a reaction cell comprising particles complexed to platelet factor 4 (PF4), which particle-complexed PF4 reacts specifically with heparin/platelet factor 4 antibodies, and said particles forming specific aggregates upon contacting heparin/platelet factor 4 antibodies; and    wherein the presence of heparin/platelet factor 4 antibodies indicates HIT in the subject.    
   
   
       97 . An apparatus for detecting heparin/platelet factor 4 antibodies in a fluid sample comprising: 
 (a) a tower comprising at least one foot and a block channel foot;    (b) an ampoule support having at least one slot for holding an ampoule and a reagent well;    (c) a cover with an opening for receiving the tower such that the at least one foot crushes a first ampoule in the ampoule support while the block channel foot blocks the flow of fluid flow to the reagent well; and    (d) a bottom plate with an indentation for holding a test strip, the bottom plate suitable for coupling to the ampoule support, which in-turn holds an ampoule and is covered by the cover;    whereby the tower is depressed into the cover to crush the first ampoule to allow a first reagent in the first ampoule to flow out while the block channel foot blocks the flow of the first reagent to the reagent well, and wherein withdrawing the tower allows the first reagent to flow to the reagent well.    
   
   
       98 . The apparatus of  claim 97 , wherein the tower further comprises a spur to crush a second ampoule.  
   
   
       99 . The apparatus of  claim 97 , wherein a second reagent in the second ampoule is mixed with the first reagent prior to flowing into the reagent well.  
   
   
       100 . The apparatus of  claim 97 , wherein the spur is adjacent to the block channel foot.  
   
   
       101 . The apparatus of  claim 97 , wherein the ampoule support comprises a channel connected to the reagent well.  
   
   
       102 . The apparatus of  claim 97 , wherein the bottom plate, the test trip, the ampoule support, and the cover are engaged to form a test device operable by depressing and withdrawing the tower.