Reagents, Systems and Methods for Analyzing White Blood Cells

Abstract

Aspects of the invention include WBC analysis reagents, systems and methods that can be used for analyzing a sample of whole blood to identify, classify, and/or quantify white blood cells (WBC) and WBC sub-populations in the sample. The WBC analysis reagents of the present disclosure generally include at least one membrane-permeable fluorescent dye, a WBC protecting reagent, and a surfactant. In some embodiments, the WBC reagents include a suitable amount of an osmolality adjusting component to adjust the osmolality of the WBC reagent into a desired range.

1 .- 21 . (canceled) 
     
     
         22 . A system for conducting a white blood cell (WBC) differential analysis on a sample of whole blood, the system comprising:
 (a) a hematology analyzer, the hematology analyzer comprising:
 an excitation source positioned to excite particles within the blood sample; 
 a plurality of detectors including (1) an axial light loss detector positioned to measure axial light loss from the excited blood sample, (2) an intermediate angle scatter detector positioned to measure intermediate angle scatters from the excited blood sample, (3) a polarized side scatter detector positioned to measure large angle polarized side scatters from the excited blood sample, (4) a depolarized side scatter detector positioned to measure large angle depolarized side scatter from the excited blood sample, and (5) a fluorescence detector positioned to measure fluorescence emitted from the excited blood sample; and 
 a processor configured to:
 (I) receive the measurements of (1) axial light loss, (2) intermediate angle scatters, (3) large angle polarized side scatters, (4) large angle depolarized side scatters, and (5) fluorescence from the plurality of detectors, and 
 (II) perform a WBC differential analysis of the blood sample, based on all five measurements, for particles that emit fluorescence above a fluorescence threshold; and 
 
   (b) a reagent for analyzing WBCs in the sample, the reagent comprising:
 a membrane-permeable fluorescent dye; and 
 an osmolality adjustment component; 
   wherein the concentration of the membrane-permeable fluorescent dye is sufficient to facilitate identification of one or more cells in the sample that contain a nucleus using the hematology analyzer; and   wherein the concentration of the osmolality adjustment component is sufficient to facilitate identification of a plurality of subpopulations of WBCs in the sample using the hematology analyzer.   
     
     
         23 . The system according to  claim 22 , wherein the membrane-permeable fluorescent dye is acridine orange, hexidium iodide, SYTO RNA Select, SYTO 12 or SYTO 14. 
     
     
         24 . The system according to  claim 22 , wherein the concentration of the membrane-permeable fluorescent dye in the reagent ranges from about 0.0001% up to about 0.0005%. 
     
     
         25 . The system according to  claim 22 , wherein the concentration of the membrane-permeable fluorescent dye in the reagent ranges from about 0.01 μM up to about 15 μM. 
     
     
         26 . The system according to  claim 22 , wherein the osmolality adjustment component is ammonium chloride or sodium chloride. 
     
     
         27 . The system according to  claim 22 , wherein the concentration of the osmolality adjustment component ranges from about 0.1% up to about 0.5%. 
     
     
         28 . The system according to  claim 22 , wherein the reagent further comprises a WBC protecting agent. 
     
     
         29 . The system according to  claim 28 , wherein the WBC protecting agent is formaldehyde, glutaraldehyde, butoxyethanol, phenoxyethanol, or isopropyl alcohol. 
     
     
         30 . The system according to  claim 28 , wherein the concentration of the WBC protecting agent ranges from about 0.1% up to about 1.0%. 
     
     
         31 . The system according to  claim 22 , wherein the reagent further comprises a surfactant. 
     
     
         32 . The system according to  claim 31 , wherein the surfactant is saponin. 
     
     
         33 . The system according to  claim 31 , wherein the concentration of the surfactant ranges from about 0.01% up to about 0.05%. 
     
     
         34 . The system according to  claim 22 , wherein the reagent further comprises a pH buffering component. 
     
     
         35 . The system according to  claim 34 , wherein the pH buffering component is sodium acetate or sodium bicarbonate. 
     
     
         36 . The system according to  claim 34 , wherein the concentration of the pH buffering component ranges from about 0.01% up to about 0.5%. 
     
     
         37 . The system according to  claim 22 , wherein the reagent further comprises an antimicrobial agent. 
     
     
         38 . The system according to  claim 37 , wherein the concentration of the antimicrobial agent ranges from about 0.01% up to about 0.1%. 
     
     
         39 . The system according to  claim 22 , wherein the pH of the reagent ranges from about 2.5 up to about 12.5 pH units. 
     
     
         40 . The system according to  claim 22 , wherein the osmolality of the reagent ranges from about 25 up to about 350 mOsm. 
     
     
         41 . The system according to  claim 22 , wherein the processor is further configured to pre-screen the received measurements to remove from consideration any particles that do not meet the fluorescence threshold. 
     
     
         42 . The system according to  claim 22 , wherein the axial light loss detector measures axial light loss at 0° scatter. 
     
     
         43 . The system according to  claim 22 , wherein the intermediate angle scatter detector measures light angle scatter at about 3° to about 15°. 
     
     
         44 . The system according to  claim 22 , wherein the plurality of detectors includes one or more photomultiplier tubes. 
     
     
         45 . The system according to  claim 22 , wherein the excitation source is a laser. 
     
     
         46 . The system according to  claim 45 , wherein the laser emits light at a wavelength corresponding to the fluorescent dye. 
     
     
         47 . The system according to  claim 22 , wherein the fluorescent dye is selected to correspond with the excitation source. 
     
     
         48 . The system according to  claim 22 , further comprising an incubation subsystem for diluting the blood sample with the reagent. 
     
     
         49 . The system according to  claim 48 , wherein the incubation subsystem is configured to incubate the blood sample with the reagent for a period of time that is less than about 25 seconds. 
     
     
         50 . The system according to  claim 48 , wherein the incubation subsystem is configured to incubate the blood sample with the reagent for a period of time that is less than about 17 seconds. 
     
     
         51 . The system according to  claim 48 , wherein the incubation subsystem is configured to incubate the blood sample with the reagent for a period of time that is less than about 9 seconds. 
     
     
         52 . The system according to  claim 48 , wherein the incubation subsystem is configured to incubate the blood sample with the reagent at a temperature ranging from about 30° C. to about 50° C. 
     
     
         53 . The system according to  claim 48 , wherein the incubation subsystem is configured to incubate the blood sample with the reagent at a temperature of about 40° C. 
     
     
         54 .- 72 . (canceled)