Apparatus and methods for rapid detection of acute phase reactants and white blood cells

Abstract

The present invention provides diagnostic devices and methods for quantifying the amounts of an acute phase reactant (e.g., C-reactive protein (CRP) or serum amyloid A (SAA)) in a body fluid sample and/or white blood cell counts in blood sample. In particular, the present invention provides a rapid assay to detect CRP, SAA, and/or white blood cells in blood with high sensitivity and specificity.

We claim: 
     
         1 . A device for assaying a targeted cell or a non-cell analyte in a blood sample, comprising:
 (a) two plates facing each other and separated by a gap, wherein each of the plates have a sample contact area for contacting a sample that contains or is suspected of containing a targeted cell and a non-cell analyte;   (b) a cell staining reagent and/or a capture agent, are coated in one or both of the sample contact area;   wherein the sample contact areas of the plates confine the sample into a thin sample layer; wherein the thin sample layer has a thickness confined by the gap, the staining reagent stains the targeted cell, and the capture agent binds the non-cell analyte; wherein the gap has a size configured to make a targeted cell have a monolayer in the thin sample layer and wherein in the monolayer, the targeted cells do not have significant overlap.   
     
     
         2 . A device for assaying a targeted cell and a non-cell analyte in a blood sample, comprising:
 (a) two plates facing each other and separated by a gap, wherein each of the plates have a sample contact area for contacting a sample that contains or is suspected of containing a targeted cell and a non-cell analyte;   (b) a microstructure that is between the sample contact areas of the two plates;   (c) a cell staining reagent and/or a capture agent coated either on the surface of the microstructure or in one or both of the sample contact area;   wherein the sample contact areas of the plates confine the sample into a thin sample layer; wherein the thin sample layer has a thickness confined by the gap, the staining reagent stains the targeted cell, and the capture agent binds specifically to the non-cell analyte; wherein the gap has a size configured to make a targeted cell having a monolayer in the thin sample layer and wherein in the monolayer, the targeted cells do not have significant overlap.   
     
     
         3 . The device of  claim 1 , further comprises an imager that takes one or more images of the sample layer for detecting and counting the blood cells, and detecting and/or quantifying the analyte. 
     
     
         4 . The device of  claim 1 , further comprising a detection agent, wherein the detection agent binds specifically the non-cell analyte. 
     
     
         5 . The device of  claim 1 , wherein the two plates are movable relative to each other, wherein the gap between the two plates are regulated by spacers, wherein at least one of the spacers is in the sample contact area. 
     
     
         6 . The device of  claim 4 , wherein the capture reagent or the detection agent is an antibody, a nucleic acid, or an aptamer. 
     
     
         7 . The device of  claim 4 , wherein the capture or detection agents are the monoclonal antibody binds to CRP or SAA. 
     
     
         8 . The device of  claim 1 , wherein the microstructure is conjugated with antibody. 
     
     
         9 . The device of  claim 1 , wherein the microstructure is a plurality of beads. 
     
     
         10 . The device of  claim 9 , wherein the plurality of beads is made of polystyrene or silica. 
     
     
         11 . The device of  claim 9 , wherein the plurality of bead has a diameter of 2 to 30 μm. 
     
     
         12 . The device of  claim 9 , wherein the plurality of bead has a diameter of 10 μm. 
     
     
         13 . The device of  claim 4 , wherein the detection reagent is an antibody. 
     
     
         14 . The device of  claim 13 , wherein the antibody is a monoclonal antibody. 
     
     
         15 . The device of  claim 13 , wherein the monoclonal antibody binds to a complex of the capture reagent and CRP, or a complex of the capture agent and SAA. 
     
     
         16 . The device of  claim 4 , wherein the detection agent is labeled with a fluorescent label. 
     
     
         17 . The device of  claim 16 , the fluorescent label is FITC, rhodamine, or Cy5. 
     
     
         18 . The device of  claim 1 , further comprising a stain reagent, wherein the stain reagent is SYTO 9 or YOYO. 
     
     
         19 . A device for assaying a blood cell and an analyte in the blood, comprising:
 (a) a first plate;   (b) a second plate;   (c) a plurality of spacers fixed to at least one of the first plate and the second plate; and   (d) a sample contact area disposed on at least one of the first plate and the second plate;   wherein the plurality of spacers have (i) a pillar shape, (ii) a substantially flat top surface, (iii) a substantially uniform height, and (iv) a constant inter-spacer distance,   wherein the sample contact area is configured to hold a blood sample that contains or is suspected of containing a first analyte and a second analyte, and   wherein the sample contact area comprises:   (i) a capture reagent on the sample contact area, wherein the capture reagent is configured to bind to the analyte to form a complex;   (ii) a detection reagent on the sample contact area, wherein the detection reagent is configured to be diffusible in the sample and bind to the complex; and   (iii) a stain reagent on the sample contact area, wherein the stain reagent is configured to stain a cell.   
     
     
         20 . The device of  claim 19 , wherein one or both of the plates of the Q-card has a plurality of spacers.