Selective ultrasonic lysis of blood and other biological fluids and tissues

Abstract

The present invention features methods for selective lysis of endogenous cells in a biological sample. In preferred embodiments, the methods of the invention comprise contacting the biological sample with lysis solution, and subjecting the mixture to ultrasound, thereby selectively lysing the endogenous cells in the biological sample. The invention also features a lysis solution comprising Saponin and Proteinase.

1 - 65 . (canceled) 
     
     
         66 . A lysis solution for selective lysis of endogenous cells in a biological sample comprising:
 a detergent; and   a proteinase.   
     
     
         67 . The lysis solution of  claim 66 , further comprising a Sodium Phosphate buffer, pH 8. 
     
     
         68 . The lysis solution of  claim 67 , wherein the Sodium Phosphate buffer is at pH 8. 
     
     
         69 . The lysis solution of  claim 66 , wherein the detergent is selected from the group consisting of Saponin, nonionic surfactant and polysorbate surfactant. 
     
     
         70 . The lysis solution of  claim 69 , wherein the nonionic surfactant comprises Triton X-100. 
     
     
         71 . The lysis solution of  claim 69 , wherein the polysorbate surfactant comprises Tween 20. 
     
     
         72 . The lysis solution of  claim 66 , wherein the detergent is Saponin. 
     
     
         73 . The lysis solution of  claim 72 , wherein the Saponin is from  Quillaja  bark. 
     
     
         74 . The lysis solution of  claim 66 , wherein the proteinase is derived from  Aspergillus.    
     
     
         75 . The lysis solution of  claim 74 , wherein the proteinase is derived from  Aspergillus melleus.    
     
     
         76 . The lysis solution of  claim 72 , comprising 0.1%-10% Saponin. 
     
     
         77 . The lysis solution of  claim 76 , comprising 1.15% Saponin. 
     
     
         78 . The lysis solution of  claim 66 , comprising 5.0-37.5 Units Proteinase. 
     
     
         79 . The lysis solution of  claim 78 , comprising 11.25 Units Proteinase. 
     
     
         80 . The lysis solution of  claim 67 , comprising 0.01-0.1M Sodium Phosphate buffer, pH 8. 
     
     
         81 . The lysis solution of  claim 80 , comprising 0.1M Sodium Phosphate buffer, pH 8. 
     
     
         82 . The lysis solution of  claim 66 , further comprises an enzyme selected from the group consisting of cholesterol esterase, lipase and DNase. 
     
     
         83 . The lysis solution of  claim 66 , further comprises a reducing agent. 
     
     
         84 . The lysis solution of  claim 83 , wherein the reducing agent comprises TCEP. 
     
     
         85 . The lysis solution of  claim 66 , further comprises a chaotropic agent. 
     
     
         86 . The lysis solution of  claim 85 , wherein the chaotropic agent comprises guanidinium chloride. 
     
     
         87 . The lysis solution of  claim 66 , further comprising a hypotonic salt solution. 
     
     
         88 . The lysis solution of  claim 66 , wherein the biological sample is a body fluid. 
     
     
         89 . The lysis solution of  claim 88 , wherein the body fluid is selected from the group consisting of: blood or blood fractions, respiratory secretions, cerebrospinal fluid, urine, stool, wound exudates (pus), and naso-pharyngeal fluid/mucus. 
     
     
         90 . The lysis solution of  claim 66 , where the biological sample is selected from the group consisting of: platelets, platelet concentrate and a mammalian cell culture. 
     
     
         91 . A lysis solution for increasing filterability of bronchoalveolar lavage comprising:
 Triton X-100 or Tween 20;   Protease; and   TCEP.   
     
     
         92 . The lysis solution of  claim 91 , further comprises Sodium Phosphate Buffer, pH 8. 
     
     
         93 . A method for selective lysis of endogenous cells in a bronchoalveolar lavage sample and increasing filterability of the sample comprising:
 contacting the bronchoalveolar lavage sample with the lysis solution of claim  26 ; and   subjecting the mixture of the bronchoalveolar lavage sample and lysis solution to high-frequency ultrasound;   thereby selectively lysing the endogenous cells in the bronchoalveolar lavage sample.   
     
     
         94 . The method of  claim 93 , wherein the endogenous cells are mammalian cells. 
     
     
         95 . The method of  claim 93 , wherein the bronchoalveolar lavage sample comprises microorganisms. 
     
     
         96 . The method of  claim 95 , wherein the microorganisms are selected from the group consisting of: bacteria, yeast and fungi. 
     
     
         97 . The method of  claim 93 , further comprising filtering or centrifuging the lysed sample; and
 detecting, identifying, characterizing or quantifying microorganisms in the biological sample;   thereby detecting, identifying, characterizing or quantifying microorganisms in the biological sample.   
     
     
         98 . The method of  claim 97 , wherein the detecting, identifying, characterizing or quantifying is carried out using peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH).