Reagents and methods for detecting pnh type ii white blood cells and their identification as risk factors for thrombotic disorders

Abstract

The disclosure relates to methods for detecting PNH Type II cell populations in biological samples as well as methods for determining whether a patient is at an increased risk for developing thrombocytopenia or thrombosis based on the percentage of PNH Type II cells in the patient's blood. The disclosure also features reagents and conjugates for use in the methods.

1 . A method for predicting whether a patient is at an increased risk for developing thrombosis, the method comprising:
 determining the percentage of PNH Type II white blood cells of the total white blood cells of the same histological type in a biological sample from a patient; and   predicting whether the patient is at an increased risk for developing thrombosis, wherein the patient is at an increased risk for developing thrombosis if the percentage of PNH Type II white blood cells is greater than or equal to 1.2%.   
     
     
         2 . The method of  claim 1 , wherein the white blood cells are i) granulocytes or ii) monocytes. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the biological sample is a whole blood sample. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein a PNH Type II white blood cell population that is between 1.2% to 65.3%, inclusive of 1.2% and 65.3%, indicates that the patient is at an increased risk for thrombosis. 
     
     
         7 . The method of  claim 1 , wherein a PNH Type II white blood cell population that is greater than or equal to a) 5%, b) 10%, c) 20%, or d) 50% indicates that the patient is at an increased risk for thrombosis. 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The method of  claim 1 , further comprising monitoring the patient for the development of at least one symptom of thrombosis if the patient is at an increased risk of developing thrombosis. 
     
     
         12 . The method of  claim 1 , further comprising selecting an anti-thrombotic therapy for the patient if the patient is at an increased risk of developing thrombosis. 
     
     
         13 . The method of  claim 12 , wherein the anti-thrombotic therapy is an anticoagulant or thrombolytic agent. 
     
     
         14 . The method of  claim 13 , wherein the anticoagulant is coumadin, heparin, or derivatives thereof. 
     
     
         15 . The method of  claim 13 , wherein the thrombolytic agent is a tissue plasminogen activator, streptokinase, or a urokinase-type plasminogen activator. 
     
     
         16 . The method of  claim 1 , further comprising administering to the patient an anti-thrombotic therapy if the patient is at an increased risk for developing thrombosis. 
     
     
         17 . A method for selecting a therapy for a patient, the method comprising: selecting one or both of an anti-thrombotic therapy and an anti-thrombocytopenic therapy for a patient determined to have a PNH Type II white blood cell population of greater than or equal to 1.2%. 
     
     
         18 . A method for treating a patient, the method comprising administering to a patient in need thereof one or both of an anti-thrombotic therapy and an anti-thrombocytopenic therapy if the patient has a PNH Type II white blood cell population of greater than 1.2%. 
     
     
         19 . The method of  claim 17  or  18 , wherein the anti-thrombotic therapy is an anticoagulant or thrombolytic agent. 
     
     
         20 . The method of  claim 17  or  18 , wherein the anti-thrombocytopenic therapy is a platelet transfusion. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The method of any one of  claim 1 ,  17 , or  18 , wherein a non-lytic variant form of aerolysin protein is used to determine the percentage of PNH Type II white blood cells. 
     
     
         24 . A method for identifying a PNH Type II white blood cell, the method comprising:
 contacting a plurality of white blood cells with a reagent that binds to: (i) GPI or (ii) a GPI-anchored protein; and   identifying one or more of the white blood cells as PNH Type II white blood cells based on the amount of reagent bound to the cells, wherein an intermediate amount of binding of the reagent to a white blood cell, as compared to the amount of binding of the reagent to a PNH Type III white blood cell and the amount of binding of the reagent to a Type I white blood cell, indicates that the white blood cell is a PNH Type II white blood cell.   
     
     
         25 . A method for distinguishing between white blood cell populations, the method comprising:
 contacting a plurality of white blood cells with a reagent that binds to: (i) GPI or (ii) a GPI-anchored protein; and   distinguishing at least a portion of the white blood cells from other white blood cells of the plurality based on the amount of reagent bound to the cells, wherein the PNH Type II white blood cells, if present, are sufficiently distinguished from the Type I white blood cells and PNH Type III white blood cells of the same histological type to allow the percentage of PNH Type II white blood cells of the total white blood cells of the same histological type in the plurality to be determined.   
     
     
         26 . The method of  claim 24  or  25 , further comprising determining the percentage of PNH
 Type II white blood cells of the total white blood cells of the same histological type in the plurality. 
 
     
     
         27 . A method for determining the percentage of PNH Type II white blood cells, the method comprising:
 providing a plurality of white blood cells contacted with a reagent that binds to: (i) GPI or (ii) a GPI-anchored protein;   distinguishing at least a portion of white blood cells from other white blood cells of the plurality based on the amount of reagent bound to the cells, wherein the PNH Type II white blood cells, if present, are sufficiently distinguished from the Type I white blood cells and PNH Type III white blood cells of the same histological type to allow the percentage of PNH Type II white blood cells of the total white blood cells of the same histological type in the plurality to be determined; and   determining the percentage of PNH Type II white blood cells.   
     
     
         28 . The method of  claim 26  or  27 , further comprising determining the percentage of PNH
 Type III white blood cells. 
 
     
     
         29 . The method of  claim 25  or  27 , wherein the distinguishing comprises flow cytometry. 
     
     
         30 . The method of any one of  claims 24 ,  25 , or  27 , wherein the plurality of white blood cells are obtained from a patient having, suspected of having, or at risk of developing PNH. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . The method of anyone of  claims 24 ,  25 , or  27 , wherein the reagent binds to a human GPI moiety. 
     
     
         34 . The method of  claim 33 , wherein the reagent comprises an aerolysin protein. 
     
     
         35 . The method of  claim 34 , wherein the reagent comprises a variant form of aerolysin protein that is non-lytic or is substantially non-lytic as compared to the wild-type form of the protein. 
     
     
         36 . The method of  claim 33 , wherein the reagent comprises the amino acid sequence depicted in SEQ ID NO:2 or 7 wherein the threonine at position 253 is substituted with a cysteine and the alanine at position 300 is substituted for a cysteine. 
     
     
         37 . The method of  claim 33 , wherein the reagent is an antibody or an antigen-binding fragment thereof. 
     
     
         38 . The method of any one of  claims 24 ,  25 , or  27 , wherein the reagent binds to a GPI-anchored protein. 
     
     
         39 . The method of  claim 38 , wherein the GPI-anchored protein is selected from the group consisting of alkaline phosphatase, 5′ nucleotidease acetylcholinesterase, dipeptidase, LFA-3, NCAM, PH-20, CD55, CD59, Thy-1, Qa-2, CD14, CD33, CD16 (the Fcγ receptor III), carcinoembryonic antigen (CEA), CD24, CD66b, CD87, CD48, and CD52. 
     
     
         40 . The method of  claim 38 , wherein the reagent is an antibody or an antigen-binding fragment thereof. 
     
     
         41 . The method of any one of  claims 24 ,  25 , or  27 , wherein the plurality of white blood cells are contacted with a reagent that bind to GPI and a reagent that binds to a GPI-linked protein.