G - Physics – 01 – N
Patent
G - Physics
01
N
150/11, 150/3
G01N 33/50 (2006.01) G01N 1/28 (2006.01) G01N 15/12 (2006.01) G01N 33/48 (2006.01)
Patent
CA 1338603
A method and reagent system are disclosed for the rapid isolation, identification and/or analysis of leukocytes from a whole blood sample. The method and reagent system of this invention has application to any environment in which the study and/or analysis of the leukocyte fraction of whole blood requires their isolation in their native or near native state. One of the environments in which this invention can be used to advantage is in the performance of white cell differentiation on automated instrumentation designed for that purpose. A second environment which is also particularly suitable for the practice of this invention is in the preparation of whole blood samples for immunological staining and subsequent flow cytometric analysis. The lytic reagent of this invention comprises a water soluble compound which at least partially dissociates in aqueous solution to release a proton and a counterion, thereby acidifying the sample to a pH in the range of from about 2.6 to 4Ø The relative concentration of the lytic reagent in the sample can range from about 0.01 to about 0.1 for relatively strong acidic compounds and from about 0.05 to about 0.5 for relatively weak acidic compounds. In each instance, the relative concentration of such compounds is carefully controlled to maintain the osmolality of the sample below about 100 mOs. The identity and compatibility of the counterion of the compound can influence the extent of differentiation of the leukocyte population attainable in subsequent analysis. Accordingly, some empirical testing may be required to obtain both optimum lysis and differentiation. The reagent system of this invention can be used to effect selective stromatolysis of red blood cells and create subtle modifications to the leukocyte population to enable their automated differentiation into five (5) sub-populations. The advantage and uniqueness of this reagent system is the surprising speed at which it is able to effect the foregoing objectives (generally less than 10 seconds at room temperature) and the ability to further differentiate the leukocyte population (notably, the granulocyte population). Following stromatolysis, a quenching agent is added to retard the reactivity of the lytic reagent system and, thus, inhibit any further dramatic changes to the leukocyte population. The treatment of the whole blood sample in the foregoing manner is further unique in that the leukocyte fraction of the sample has retained its characteristic immunochemical response.
561207
Crews Harold R.
Fischer Timothy J.
Ledis Stephen L.
Sena Ted
Borden Ladner Gervais Llp
Coulter International Corp.
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