Software Integrated Flow Cytometric Assay For Quantification Of The Human Polymorphonuclear Leukocyte FCgammaRI Receptor (CD64)

Abstract

A composition for evaluating a biological condition is disclosed. The composition has (a) a sample composition comprising at least one of: i.) a bodily specimen comprising a target moiety; ii.) a positive control moiety; and iii.) a negative control moiety; (b) an antibody composition comprising at least one of: i.) at least one target antibody; ii.) at least one positive control identifying antibody; and iii.) at least one negative control identifying antibody; and (c) at least one reference composition comprising at least one of: i.) a target signal reference composition; and ii.) a reference identifier composition.

We claim: 
     
         1 . A composition for evaluating a biological condition, the composition comprising:
 a. a sample composition comprising at least one of:
 i. a bodily specimen comprising a target moiety; 
 ii. a positive control moiety; and 
 iii. a negative control moiety; 
   b. an antibody composition comprising at least one of:
 i. at least one target antibody; 
 ii. at least one positive control identifying antibody; and 
 iii. at least one negative control identifying antibody; and 
   c. at least one reference composition comprising at least one of:
 i. a target signal reference composition; and 
 ii. a reference identifier composition. 
   
     
     
         2 . A composition according to  claim 1 , further comprising at least one conditioning moiety comprising:
 a. at least one lysis reagent; and   b. at least one diluent.   
     
     
         3 . A composition according to  claim 2 , wherein the biological condition is selected from a group consisting of bacterial infection and sepsis. 
     
     
         4 . A composition according to  claim 3 , wherein the bodily specimen comprises blood. 
     
     
         5 . A composition according to  claim 4 , wherein said target moiety comprises a CD64 surface antigen on neutrophils. 
     
     
         6 . A composition according to  claim 5 , wherein said positive control moiety comprises monocytes and said negative control comprises lymphocytes. 
     
     
         7 . A composition according to  claim 6 , wherein the target moiety is CD64 on neutrophils, the positive control moiety comprises CD64 expression on monocytes, and the negative control moiety comprises lymphocytes without CD64 expression. 
     
     
         8 . A composition according to  claim 5 , further comprising a signaling moiety on said at least one target antibody. 
     
     
         9 . A composition according to  claim 1 , wherein at least one reference composition comprises beads. 
     
     
         10 . A composition according to  claim 9 , wherein said beads comprise polystyrene microbeads. 
     
     
         11 . A composition according to  claim 1 , wherein the target signal reference composition comprises a first fluorescent signal and said reference identifier composition comprises a second fluorescent signal. 
     
     
         12 . A composition according to  claim 11 , wherein said first fluorescent signal comprises FITC and said second fluorescent signal comprises Starfire Red fluor. 
     
     
         13 . A method for quantifying a biomarker in a sample, comprising:
 a. contacting the sample with a fluorescently-labeled binding moiety that specifically binds to the biomarker to form a labeled sample;   b. detecting a first fluorescent signal from at least a portion of the labeled sample;   c. detecting a second fluorescent signal from a population of fluorescently-labeled particles, wherein the population comprises a known fluorescent intensity; and   d. normalizing the first fluorescent signal to the second fluorescent signal, thereby quantifying the biomarker, wherein the normalizing comprises using a device comprising software capable of comparing the first and second fluorescent signal.   
     
     
         14 . The method of  claim 13 , wherein the biomarker is a sepsis biomarker. 
     
     
         15 . The method of  claim 13 , wherein the biomarker is CD64 or CD163. 
     
     
         16 . The method of  claim 13 , wherein the sample is a blood sample. 
     
     
         17 . The method of  claim 13 , wherein the first fluorescent signal and the second fluorescent signal comprise the same fluor. 
     
     
         18 . The method of  claim 13 , wherein the binding moiety is an antibody. 
     
     
         19 . The method of  claim 13 , wherein the software is capable of recognizing a specific lot of fluorescently-labeled particles. 
     
     
         20 . The method of  claim 19 , wherein said specific lot of fluorescently-labeled particles comprise at least said first fluorescent signal and said second fluorescent signal. 
     
     
         21 . The method of  claim 13 , wherein said fluorescently-labeled binding moiety targets a first cell population and a second cell population in the sample. 
     
     
         22 . The method of  claim 21 , wherein detection of binding of the binding moiety to the second cell population provides an internal positive control for the sample. 
     
     
         23 . The method of  claim 22 , wherein the binding moiety is anti-CD64 antibody and the first cell population comprises polymorphonuclear leukocytes. 
     
     
         24 . The method of  claim 23 , wherein the second cell population comprises monocytes. 
     
     
         25 . The method of  claim 13 , further comprising the step of determining the presence of at least one cell population in the sample that is not bound by the binding moiety, thus providing an internal negative control for the sample.