Flow Cytometry Reagent System

Abstract

A method of measuring cellular hemoglobin of a blood sample includes mixing a blood sample with a permeation reagent, and incubating the sample mixture to permeate cellular membrane of red blood cells and to cause hemoglobin aggregation within the cells; adding a neutralization reagent to inhibit further reactions of the permeation reagent; performing a cell-by-cell measurement of side scatter signals of the red blood cells in the sample mixture on a flow cytometer; and obtaining cellular hemoglobin (Hgb cell ) of each red blood cell using the obtained side scatter signals. The method further includes measuring cellular hemoglobin of reticulocytes (Hgb retic ) by differentiating reticulocytes using a simultaneous fluorescence measurement. The method also includes measuring cellular percentage of a hemoglobin variant in mature red blood cells or reticulocytes by adding a fluorescent antibody in the neutralization reagent and detecting fluorescence signals of antibody bound hemoglobin variant.

1 . A flow cytometry reagent system comprising:
 (a) an aqueous permeation reagent comprising:   (i) an effective amount of a N-acyl sarcosine or a salt thereof represented by following molecular structure:
   R 1 —CO—N(CH 3 )CH 2 COOX 1    
   wherein R 1  is an alkyl or alkylene group having 8 to 18 carbon atoms, and X 1  is H, Na + , or K + ; and   (ii) saccharide or glycerol;   said permeation reagent having a pH between 4 and 6 and a low ionic strength defined by a conductivity of less than 9.0 mS/cm; and   (b) a neutralization reagent comprising a buffer and an osmolality adjusting agent; said neutralization reagent being hypertonic and neutral.   
   
   
       2 . The reagent system of  claim 1 , wherein said permeation reagent further comprises a buffer. 
   
   
       3 . The reagent system of  claim 2 , wherein said buffer of said permeation reagent comprises succinic acid. 
   
   
       4 . The reagent system of  claim 1 , wherein said permeation reagent has a conductivity of less than 1.2 mS/cm. 
   
   
       5 . The reagent system of  claim 1 , wherein said permeation reagent has an osmolality from about 240 to about 280 mOsm/kg H 2 O. 
   
   
       6 . The reagent system of  claim 1 , wherein said N-acyl sarcosine is N-lauroyl sarcosine. 
   
   
       7 . The reagent system of  claim 6 , wherein said N-lauroyl sarcosine is in a concentration range from about 0.1 mM to about 10 mM. 
   
   
       8 . The reagent system of  claim 1 , wherein said saccharide is disaccharide, or monosaccharide. 
   
   
       10 . The reagent system of  claim 1 , wherein said permeation reagent further comprises albumin. 
   
   
       11 . The reagent system of  claim 10 , wherein said permeation reagent comprises bovine serum albumin. 
   
   
       12 . The reagent system of  claim 1 , wherein said osmolality adjusting agent of said neutralization reagent comprises one or more alkaline metal salt. 
   
   
       13 . The reagent system of  claim 1 , wherein said neutralization reagent has an osmolality from about 800 to about 1,200 mOsm/kg H 2 O. 
   
   
       14 . The reagent system of  claim 1 , wherein said neutralization reagent has a pH from about 7.1 to about 7.5. 
   
   
       15 . The reagent system of  claim 1 , wherein said neutralization reagent further comprises albumin. 
   
   
       16 . The reagent system of  claim 15 , wherein said neutralization reagent comprises bovine serum albumin in a concentration from about 0.4 to about 1.2 mM. 
   
   
       17 . The reagent system of  claim 1 , wherein said neutralization reagent further comprises a monoclonal antibody. 
   
   
       18 . The reagent system of  claim 1  further comprising a fixation reagent. 
   
   
       19 . The reagent system of  claim 18 , wherein said fixation reagent comprises a fixative, an osmolality adjusting agent, and a buffer. 
   
   
       20 . The reagent system of  claim 19 , wherein said fixation reagent further comprises a chelating agent.