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Publication Number
US-10184950-B2
Publication Date
2019-01-22
Expiration Date
Abstract
Methods of testing HIV viral load are described. The methods comprise detecting HIV viral RNA in a sample of leukocyte-depleted blood. Such methods can be carried out on low-volume samples obtained without the need for venipuncture or a centrifuge. The methods are particularly suited for HIV viral load testing in resource-limited settings. Methods for monitoring HIV infection are also described, as well as kits for carrying out the methods.
Core Innovation
The invention provides a method of testing HIV viral load by detecting HIV viral RNA directly in a sample of leukocyte-depleted blood without preparing a plasma sample. The sample is a whole blood sample selectively depleted of leukocytes or a diluted whole blood sample selectively depleted of leukocytes.
The approach includes selectively depleting leukocytes from whole blood or diluted whole blood to provide leukocyte-depleted blood suitable for downstream HIV viral RNA detection. The disclosed requirements and processing define leukocyte-depleted blood achieved by leukoreduction, and support use of low-volume blood sampling such as finger prick and heel prick collection.
After providing leukocyte-depleted blood, the method detects HIV viral RNA and can determine the number of copies of HIV viral RNA per milliliter of the leukocyte-depleted blood sample. The disclosed detection is based on nucleic acid detection workflows including reverse transcription with isothermal nucleic acid amplification of the reverse-transcription product, and the document further describes cut-off based and/or quantitative viral load interpretation.
Claims Coverage
The document includes five independent claims that collectively cover plasma-free HIV viral RNA detection in leukocyte-depleted whole blood or diluted leukocyte-depleted whole blood, with additional refinements on low-volume sampling, isotonic dilution, and dried-sample rehydration. The inventive feature set centers on leukocyte-depleted blood for HIV viral load testing without plasma preparation.
Detecting HIV viral RNA in leukocyte-depleted blood without plasma
Detecting HIV viral RNA in a sample of leukocyte-depleted blood, wherein the sample is a whole blood sample selectively depleted of leukocytes or a diluted whole blood sample selectively depleted of leukocytes, and wherein the method is carried out without preparing a plasma sample.
Low-volume leukocyte-depleted whole blood without plasma
Selectively depleting a whole blood sample, or a diluted whole blood sample, of leukocytes to provide a sample of leukocyte-depleted blood, wherein the whole blood sample is a low volume whole blood sample up to 500 μl; detecting HIV viral RNA in the sample of leukocyte-depleted blood; and wherein the method is carried out without preparing a plasma sample.
Isotonic dilution of diluted leukocyte-depleted whole blood without plasma
Selectively depleting a whole blood sample, or a diluted whole blood sample, of leukocytes to provide a sample of leukocyte-depleted blood, wherein the diluted whole blood sample is diluted at least 1-in-2 with an isotonic solution; detecting HIV viral RNA in the sample of leukocyte-depleted blood; and wherein the method is carried out without preparing a plasma sample.
Rehydrating a dried leukocyte-depleted blood sample without plasma
Rehydrating a dried sample of leukocyte-depleted blood to provide a sample of leukocyte-depleted blood, wherein the dried sample is a whole blood sample selectively depleted of leukocytes or a diluted whole blood sample selectively depleted of leukocytes; detecting HIV viral RNA in the rehydrated sample of leukocyte-depleted blood; and wherein the method is carried out without preparing a plasma sample.
Drying then rehydrating leukocyte-depleted blood without plasma
Drying a sample of leukocyte-depleted blood, wherein the sample is a whole blood sample selectively depleted of leukocytes or a diluted whole blood sample selectively depleted of leukocytes; rehydrating the dried sample; and detecting HIV viral RNA in the rehydrated sample, wherein the method is carried out without preparing a plasma sample.
Across the independent claims, the core claim coverage is directed to plasma-free testing of HIV viral load by detecting HIV viral RNA in leukocyte-depleted blood. The claim set additionally covers low-volume whole blood, dilution with an isotonic solution, and dried leukocyte-depleted blood with rehydration prior to HIV viral RNA detection.
Stated Advantages
Allows HIV viral load testing by detecting HIV viral RNA in leukocyte-depleted blood without preparing a plasma sample.
Enables low-volume whole blood sampling, including low volume whole blood sample up to 500 μl and collection by finger prick or heel prick.
Supports dried-sample handling by drying leukocyte-depleted blood and rehydrating prior to HIV viral RNA detection.
Documented Applications
Testing HIV viral load using leukocyte-depleted whole blood (whole blood or diluted whole blood selectively depleted of leukocytes) with HIV viral RNA detection without preparing plasma.
Testing HIV viral load using low-volume whole blood samples (up to 500 μl) including collection from a subject by finger prick or heel prick.
Testing HIV viral load using diluted whole blood diluted at least 1-in-2 with an isotonic solution prior to leukocyte depletion and HIV viral RNA detection without plasma preparation.
Testing HIV viral load using a dried sample of leukocyte-depleted blood that is rehydrated prior to HIV viral RNA detection without preparing plasma.
Testing HIV viral load using leukocyte-depleted blood that is dried, rehydrated, and then used for HIV viral RNA detection without preparing plasma.
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