Patent Issued for Immune profiling using small volume blood samples (USPTO 11739369): California Institute of Technology
2023 SEP 18 (NewsRx) -- By a
Patent number 11739369 is assigned to
The following quote was obtained by the news editors from the background information supplied by the inventors:
“Field
“This disclosure relates generally to the field of molecular biology, and more particularly to the use of small volume of blood samples for immune profiling.
“Background
“Increasing evidence implicates the immune system in an overwhelming number of diseases, and distinct cell types play specific roles in their pathogenesis. Studies of peripheral blood have uncovered a wealth of associations between gene expression, environmental factors, disease risk, and therapeutic efficacy. For example, in rheumatoid arthritis, multiple mechanistic paths have been found that lead to disease, and gene expression of specific immune cell types can be used as a predictor of therapeutic non-response. Furthermore, vaccines, drugs, and chemotherapy have been shown to yield different efficacy based on time of administration, and such findings have been linked to the time-dependence of gene expression in downstream pathways. However, human immune studies of gene expression between individuals and across time remain limited to a few cell types or time points per subject, constraining our understanding of how networks of heterogeneous cells making up each individual’s immune system respond to adverse events and change over time. There is a need for cost effective, easy-to-access, and non-invasive methods for immune profiling.”
In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors’ summary information for this patent: “Disclosed herein include embodiments of a method for single cell ribonucleic acid sequencing. In some embodiments, the method comprises providing a first low volume, capillary blood sample (or any low volume blood sample and/or any blood sample not obtained from a vein or by venipuncture) obtained from a subject at a first time point. The method can comprise diluting the first sample to obtain a first diluted sample. The method can comprise isolating first capillary peripheral blood mononuclear cells (cPBMCs) from the first diluted sample with gradient centrifugation. The method can comprise performing single cell ribonucleic acid sequencing (scRNA-seq) on the first cPBMCs isolated to generate first scRNA-seq data. The method can comprise determining a first scRNA profile of the subject at the first time point using the first scRNA-seq data and single-nucleotide polymorphisms (SNPs) of the subject.
“In some embodiments, the method comprises providing a second low volume, capillary blood sample obtained from a subject at a second time point. The method can comprise diluting the second sample to obtain a second diluted sample. The method can comprise isolating second cPBMCs from the second diluted sample with gradient centrifugation. The method can comprise performing scRNA-seq on the second cPBMCs isolated to generate second scRNA-seq data. The method can comprise determining a second scRNA profile of the subject at the second time point using the second scRNA-seq data and SNPs of the subject.
“In some embodiments, the first time point and the second time point are about 2 hours to about 24 hours apart. In some embodiments, the subject is in a first health state at the first time point, and the subject is in a second health state at the second time point. The first health state at the first time point can comprise a first disease state of a disease, and the second health state at the second time point can comprise a second disease state of the disease. The first health state at the first time point can comprise first symptoms, and the second health state at the second time point can comprise second symptoms. The first symptoms and the second symptoms can be identical, the first symptoms and the second symptoms can be different, the first symptoms can comprise the second symptoms, and/or the second symptoms can comprise the first symptoms. The first symptoms and the second symptoms can comprise an identical symptom of different severities. In some embodiments, the method comprises receiving the first health state of the subject at the first time point and the second health state of the subject at the second time point. In some embodiments, the method comprises correlating the first health state of the subject at the first time point with the first scRNA profile of the subject at the first time point. The method can comprise correlating the second health state of the subject at the second time point with the second scRNA profile of the subject at the second time point.
“In some embodiments, the method comprises determining a difference between the scRNA profile of the subject at the first time point and the second scRNA profile of the subject at the second time point. The method can thereby determine one or more genes of interest. The one or more genes of interest can comprise diurnal genes. The one or more genes of interest can comprise one or more genes each with a time of day variation in the first scRNA profile and the second scRNA profile. The method can comprise designing a gene panel comprising the one or more genes of interest. The method can comprise determining a difference between the first health state of the subject at the first time point and the second health state of the subject at the second time point. In some embodiments, the method comprises correlating (i) the difference between the scRNA profile of the subject at the first time point and the second scRNA profile of the subject at the second time point and (ii) the difference between the first health state of the subject at the first time point and the second health state of the subject at the second time point.
“In some embodiments, said determining comprises: performing sample demultiplexing of the first scRNA data of the subject and/or the second scRNA data of the subject using SNPs of the subject to determine the first scRNA profile of the subject and/or the second scRNA profile of the subject. In some embodiments, performing sample demultiplexing of the first scRNA data of the subject comprises: classifying scRNA-seq reads with an identical cell label in the first scRNA data as reads generated from a cell of a sample obtained from the subject based on (i) SNPs present in one or more of the scRNA-seq reads with the identical cell label and, (ii) optionally, SNPs of the subject. In some embodiments, performing the sample demultiplexing of the first scRNA data of the subject comprises: classifying scRNA-seq reads with an identical cell label in the second scRNA data as reads generated from a cell of a sample obtained from the subject based on SNPs present in one or more of the scRNA-seq reads with the identical cell label and (ii) optionally, SNPs of the subject. The SNPs of the subject can be determined using the first low volume, capillary blood sample of the subject. In some embodiments, the SNPs of the subject are determined by bulk RNA sequencing and/or scRNA sequencing. Said bulk RNA sequencing and/or scRNA sequencing can be performed using a low volume, capillary blood sample of the subject.”
The claims supplied by the inventors are:
“1. A method for single cell ribonucleic acid sequencing comprising: providing a first capillary blood sample obtained from a subject at a first time point; diluting the first sample to obtain a first diluted sample; isolating first capillary peripheral blood mononuclear cells (cPBMCs) from the first diluted sample with gradient centrifugation; performing single cell ribonucleic acid sequencing (scRNA-seq) on the first cPBMCs isolated to generate first scRNA-seq data; and determining a first scRNA profile of the subject at the first time point using the first scRNA-seq data and single-nucleotide polymorphisms (SNPs) of the subject.
“2. The method of claim 1, comprising: providing a second capillary blood sample obtained from the subject at a second time point; diluting the second sample to obtain a second diluted sample; isolating second cPBMCs from the second diluted sample with gradient centrifugation; performing scRNA-seq on the second cPBMCs isolated to generate second scRNA-seq data; and determining a second scRNA profile of the subject at the second time point using the second scRNA-seq data and SNPs of the subject.
“3. The method of claim 2, wherein the first time point and the second time point are about 2 hours to about 24 hours apart.
“4. The method of claim 2, wherein the subject is in a first health state at the first time point, and wherein the subject is in a second health state at the second time point.
“5. The method of claim 4, wherein the first health state at the first time point comprises a first disease state of a disease and the second health state at the second time point comprises a second disease state of the disease, wherein the first health state at the first time point comprises first symptoms and the second health state at the second time point comprises second symptoms, wherein the first symptoms and the second symptoms are identical, the first symptoms and the second symptoms are different, the first symptoms comprise the second symptoms, and/or the second symptoms comprise the first symptoms, and/or wherein the first symptoms and the second symptoms comprise an identical symptom of different severities.
“6. The method of claim 4, comprising: correlating the first health state of the subject at the first time point with the first scRNA profile of the subject at the first time point; and/or correlating the second health state of the subject at the second time point with the second scRNA profile of the subject at the second time point.
“7. The method of claim 4, comprising: determining a difference between the scRNA profile of the subject at the first time point and the second scRNA profile of the subject at the second time point, optionally thereby determining one or more genes of interest, optionally wherein the one or more genes of interest comprise diurnal genes and/or one or more genes each with a time of day variation in the first scRNA profile and the second scRNA profile; designing a gene panel comprising the one or more genes of interest; and/or determining a difference between the first health state of the subject at the first time point and the second health state of the subject at the second time point.
“8. The method of claim 7, comprising: correlating (i) the difference between the scRNA profile of the subject at the first time point and the second scRNA profile of the subject at the second time point and (ii) the difference between the first health state of the subject at the first time point and the second health state of the subject at the second time point.
“9. The method of claim 2, wherein said determining comprises: performing sample demultiplexing of the first scRNA data of the subject and/or the second scRNA data of the subject using SNPs of the subject to determine the first scRNA profile of the subject and/or the second scRNA profile of the subject.
“10. The method of claim 1, wherein the scRNA-seq comprises a whole transcriptome scRNA-seq, and wherein the scRNA profile comprises a whole transcriptome profile.
“11. The method of claim 1, wherein the first sample has a volume of about 20 ml to about 500 ml.
“12. The method of claim 1, wherein the first sample is collected by the first subject.
“13. The method of claim 1, wherein the first sample is collected in a non-clinical setting and/or out of clinic.
“14. The method of claim 1, wherein the first sample is collected using a device comprising microneedles, a device comprising microfluidic channels, a push-button collection device, or a combination thereof.
“15. The method of claim 1, wherein said diluting comprises a 1:2 to 1:50 dilution.
“16. The method of claim 1, wherein the scRNA-seq comprises a target scRNA-seq, and wherein the scRNA profile comprises expression information of a plurality of at most 1,000 genes.
“17. The method of claim 2, wherein the first sample and/or the second sample is collected from a deltoid or a finger of the subject at the first time point and/or a deltoid or a finger of the subject at the second time point.
“18. The method of claim 2, wherein said diluting comprises diluting the first sample and/or the second sample having a volume of about 100 ml to about 1 ml.
“19. The method of claim 1, wherein said isolating comprises isolating the first cPBMCs with gradient centrifugation using a density medium with a density of about 1 g/ml to about 1.5 g/ml, wherein a duration of the density centrifugation is about 10 mins to about 30 mins, and/or wherein a speed of the density centrifugation is about 500 RPM to about 1500 RPM.
“20. The method of claim 2, wherein the second sample has a volume of about 20 ml to about 500 ml.”
URL and more information on this patent, see: Brown, David. Immune profiling using small volume blood samples.
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