Microscopic detection of mononuclear cell blasts

Abstract

The present disclosure provides methods for determining the prognosis of cancer and / or infectious disease in a subject based on the number of viable mononuclear blast cells and / or the number of viable large granular lymphocytes in a blood sample obtained from the subject. TIFF2025506852000002.tif94128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 313,047, filed February 23, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present disclosure relates to methods for assessing the prognosis of cancer and / or infectious diseases in a subject. [Background technology]

[0003] background Determining the prognosis of cancer and infectious diseases in an individual can be crucial to the successful development of treatment and / or screening strategies for the individual.Therefore, there is a need in the art to develop prognostic assays for the management, treatment, and screening of cancer and infectious diseases in subjects. Summary of the Invention

[0004] overview Provided herein are methods of determining a prognosis of cancer or an infectious disease in a subject, the methods comprising: (a) contacting a blood sample obtained from the subject with a cell viability dye; (b) after step (a), using the blood sample to prepare a microscope slide; (c) identifying the number of viable blast-transformed mononuclear cells and / or the number of viable large granular lymphocytes in the microscope slide using microscopic analysis; and (d) identifying a subject having an increased number of viable blast-transformed mononuclear cells and / or an increased number of viable large granular lymphocytes compared to a reference level as having an improved prognosis of cancer or infectious disease, or identifying a subject having a decreased number of viable blast-transformed mononuclear cells and / or a decreased number of viable large granular lymphocytes compared to the reference level as having a poor prognosis of cancer or infectious disease.

[0005] In some embodiments of any of the methods described herein, step (b) comprises one or more centrifugation steps.In some embodiments of any of the methods described herein, one of the centrifugation steps comprises using a Cytospin device.In some embodiments of any of the methods described herein, the microscope slide is a permanent microscope slide that comprises a cover slip.

[0006] In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable mononuclear blast cells in the microscope slide. In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable large granular lymphocytes in the microscope slide. In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable mononuclear blast cells and the number of viable large granular lymphocytes in the microscope slide.

[0007] In some embodiments of any of the methods described herein, the reference level is the 75th percentile of the median level of the number of viable mononuclear blast cells or the 75th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.In some embodiments or any of the methods described herein, the reference level is the 80th percentile of the median level of the number of viable mononuclear blast cells or the 80th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.In some embodiments of any of the methods described herein, the reference level is the 90th percentile of the median level of the number of viable mononuclear blast cells or the 90th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.

[0008] In some embodiments of any of the methods described herein, step (b) comprises the use of one or more fluorophore-labeled antibodies specific for one or more cell surface proteins. In some embodiments of any of the methods described herein, the one or more labeled antibodies specifically bind to an antigen selected from the group consisting of CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD25, CD27, CD28, CD38, CD39, CD45, CD45RA, CD45RO, CD56, CD57, CD62L, CD66b, CD94, CD103, CD122, CD123, CD127, CD161, CD223, CD294, CCR4, CCR6, CCR7, CXCR3, CXCR5, IgA, IgD, IgE, IgG, IgM, TCRγδ, KIR, NKG2A, NKGD, NKp30, NKp44, NKp46, NKp80, CTLA-4, and GITR. In some embodiments of any of the methods described herein, the one or more labeled antibodies are one or more fluorophore-labeled antibodies.

[0009] In some embodiments of any of the methods described herein, prior to step (b), the method further comprises incubating the blood sample with an antigen specific for the infection for 6 hours to 3 days. In some embodiments of any of the methods described herein, the blood sample is incubated with the antigen for about 36 hours to about 60 hours.

[0010] In some embodiments of any of the methods described herein, the viable mononuclear cell blasts are viable T cell blasts and / or viable B cell blasts. In some embodiments of any of the methods described herein, the viable large granular lymphocytes are NK cells.

[0011] In some aspects of any of the methods described herein, the blood sample is a venous blood sample. In some aspects of any of the methods described herein, the blood sample is a capillary blood sample. In some aspects of any of the methods described herein, the capillary blood sample is a fingerstick blood sample.

[0012] Some embodiments of any of the methods described herein further comprise making a frozen blood sample from a portion of the blood sample. In some embodiments of any of the methods described herein, the blood sample is frozen in a solution comprising DMSO. In some embodiments of any of the methods described herein, the method comprises storing the frozen blood sample at a temperature below 0°C for at least 1 day. In some embodiments of any of the methods described herein, the frozen blood sample is stored at a temperature below -80°C for at least 1 day. In some embodiments of any of the methods described herein, the frozen blood sample is stored at a temperature below -190°C for at least 1 day.

[0013] Some embodiments of any of the methods described herein further include thawing the frozen blood sample and performing an additional immunological test / analysis on the thawed blood sample.

[0014] In some embodiments of any of the methods described herein, the subject has not been identified or diagnosed as having cancer or an infectious disease. In some embodiments of any of the methods described herein, the subject has previously been identified or diagnosed as having cancer or an infectious disease.

[0015] In some embodiments of any of the methods described herein, the method includes a step of selecting, for subjects identified as having an improved prognosis of cancer or infectious disease, for low frequency screening of the subjects for cancer or infectious disease.

[0016] In some embodiments of any of the methods described herein, the method includes a step of selecting, for subjects identified as having a poor prognosis for cancer or an infectious disease, for frequent screening of the subject for cancer or an infectious disease.

[0017] Also provided herein is a method of selecting a treatment for a subject who has not been identified or diagnosed as having cancer or an infectious disease, comprising the steps of: (a) contacting a blood sample obtained from the subject with a cell viability dye; (b) after step (a), using the blood sample to prepare a microscope slide; (c) identifying the number of viable mononuclear blasts and / or the number of viable large granular lymphocytes in the microscope slide using microscopic analysis; (d) identifying a subject having an increased number of viable mononuclear blasts and / or an increased number of viable large granular lymphocytes compared to a reference level as a candidate for cancer or an infectious disease. and (e) for a subject identified as having an improved prognosis of cancer or infection, selecting for less frequent screening of the subject for cancer or infection, or for a subject identified as having a poor prognosis of cancer or infection, selecting for more frequent screening of the subject for cancer or infection.

[0018] In some embodiments of any of the methods described herein, step (b) comprises one or more centrifugation steps.In some embodiments of any of the methods described herein, one of the centrifugation steps comprises the use of a Cytospin device.In some embodiments of any of the methods described herein, the microscope slide is a permanent microscope slide with a cover slip.

[0019] In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable mononuclear blast cells in the microscope slide. In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable large granular lymphocytes in the microscope slide. In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable mononuclear blast cells and the number of viable large granular lymphocytes in the microscope slide.

[0020] In some embodiments of any of the methods described herein, the reference level is the 75th percentile of the median level of the number of viable mononuclear blast cells or the 75th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.In some embodiments of any of the methods described herein, the reference level is the 80th percentile of the median level of the number of viable mononuclear blast cells or the 80th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.In some embodiments of any of the methods described herein, the reference level is the 90th percentile of the median level of the number of viable mononuclear blast cells or the 90th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.

[0021] In some embodiments of any of the methods described herein, step (b) comprises the use of one or more fluorophore-labeled antibodies specific for one or more cell surface proteins. In some embodiments of any of the methods described herein, the one or more labeled antibodies specifically bind to an antigen selected from the group consisting of CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD25, CD27, CD28, CD38, CD39, CD45, CD45RA, CD45RO, CD56, CD57, CD62L, CD66b, CD94, CD103, CD122, CD123, CD127, CD161, CD223, CD294, CCR4, CCR6, CCR7, CXCR3, CXCR5, HLA-DR, IgA, IgD, IgE, IgG, IgM, TCRγδ, KIR, NKG2A, NKGD, NKp30, NKp44, NKp46, NKp80, CTLA-4, and GITR. In some embodiments of any of the methods described herein, the one or more labeled antibodies are one or more fluorophore-labeled antibodies.

[0022] In some embodiments of any of the methods described herein, prior to step (b), the method further comprises incubating the blood sample with an antigen specific for the infection for 6 hours to 3 days. In some embodiments of any of the methods described herein, the blood sample is incubated with the antigen for about 36 hours to about 60 hours.

[0023] In some embodiments of any of the methods described herein, the viable mononuclear cell blasts are viable T cell blasts and / or viable B cell blasts. In some embodiments of any of the methods described herein, the viable large granular lymphocytes are NK cells.

[0024] In some aspects of any of the methods described herein, the blood sample is a venous blood sample. In some aspects of any of the methods described herein, the blood sample is a capillary blood sample. In some aspects of any of the methods described herein, the capillary blood sample is a fingerstick blood sample.

[0025] Some embodiments of any of the methods described herein further comprise making a frozen blood sample from a portion of the blood sample. In some embodiments of any of the methods described herein, the blood sample is frozen in a solution comprising DMSO. In some embodiments of any of the methods described herein, the method comprises storing the frozen blood sample at a temperature below 0°C for at least 1 day. In some embodiments of any of the methods described herein, the frozen blood sample is stored at a temperature below -80°C for at least 1 day. In some embodiments of any of the methods described herein, the frozen blood sample is stored at a temperature below -190°C for at least 1 day.

[0026] Some embodiments of any of the methods described herein further include thawing the frozen blood sample and performing an additional immunological test / analysis on the thawed blood sample.

[0027] In some embodiments of any of the methods described herein, the subject is identified as having an improved prognosis for cancer or infectious disease, and the subject is selected for low frequency screening for cancer or infectious disease. In some embodiments of any of the methods described herein, the subject is identified as having a poor prognosis for cancer or infectious disease, and the subject is selected for high frequency screening for cancer or infectious disease.

[0028] Also provided herein is a method of treating a subject not identified as having cancer or an infectious disease, comprising: (a) contacting a blood sample obtained from the subject with a cell viability dye; (b) after step (a), using the blood sample to prepare a microscope slide; (c) identifying the number of viable mononuclear blasts and / or the number of viable large granular lymphocytes in the microscope slide using microscopic analysis; (d) identifying subjects having an increased number of viable mononuclear blasts and / or an increased number of viable large granular lymphocytes compared to a reference level as having an improved prognosis for cancer or infectious disease, or identifying subjects having a decreased number of viable mononuclear blasts and / or a decreased number of viable large granular lymphocytes compared to a reference level as having a poor prognosis for cancer or infectious disease; and (e) performing low-frequency screening for cancer or infectious disease on the subjects identified as having an improved prognosis for cancer or infectious disease, or performing high-frequency screening for cancer or infectious disease on the subjects identified as having a poor prognosis for cancer or infectious disease.

[0029] In some embodiments of any of the methods described herein, step (b) comprises one or more centrifugation steps.In some embodiments of any of the methods described herein, one of the centrifugation steps comprises using a Cytospin device.In some embodiments of any of the methods described herein, the microscope slide is a permanent microscope slide that comprises a cover slip.

[0030] In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable mononuclear blast cells in the microscope slide. In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable large granular lymphocytes in the microscope slide. In some embodiments of any of the methods described herein, step (c) comprises determining the number of viable mononuclear blast cells and the number of viable large granular lymphocytes in the microscope slide.

[0031] In some embodiments of any of the methods described herein, the reference level is the 75th percentile of the median level of the number of viable mononuclear blast cells or the 75th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.In some embodiments of any of the methods described herein, the reference level is the 80th percentile of the median level of the number of viable mononuclear blast cells or the 80th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.In some embodiments of any of the methods described herein, the reference level is the 90th percentile of the median level of the number of viable mononuclear blast cells or the 90th percentile of the median level of the number of viable large granular lymphocytes in a healthy patient population.

[0032] In some embodiments of any of the methods described herein, step (b) comprises the use of one or more fluorophore-labeled antibodies specific for one or more cell surface proteins. In some embodiments of any of the methods described herein, the one or more labeled antibodies specifically bind to an antigen selected from the group consisting of CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD25, CD27, CD28, CD38, CD39, CD45, CD45RA, CD45RO, CD56, CD57, CD62L, CD66b, CD94, CD103, CD122, CD123, CD127, CD161, CD223, CD294, CCR4, CCR6, CCR7, CXCR3, CXCR5, HLA-DR, IgA, IgD, IgE, IgG, IgM, TCRγδ, KIR, NKG2A, NKGD, NKp30, NKp44, NKp46, NKp80, CTLA-4, and GITR. In some embodiments of any of the methods described herein, the one or more labeled antibodies are one or more fluorophore-labeled antibodies.

[0033] In some embodiments of any of the methods described herein, prior to step (b), the method further comprises incubating the blood sample with an antigen specific for the infection for 6 hours to 3 days. In some embodiments of any of the methods described herein, the blood sample is incubated with the antigen for about 36 hours to about 60 hours.

[0034] In some embodiments of any of the methods described herein, the viable mononuclear cell blasts are viable T cell blasts and / or viable B cell blasts. In some embodiments of any of the methods described herein, the viable large granular lymphocytes are NK cells.

[0035] In some aspects of any of the methods described herein, the blood sample is a venous blood sample. In some aspects of any of the methods described herein, the blood sample is a capillary blood sample. In some aspects of any of the methods described herein, the capillary blood sample is a fingerstick blood sample.

[0036] Some embodiments of any of the methods described herein further comprise making a frozen blood sample from a portion of the blood sample. In some embodiments of any of the methods described herein, the blood sample is frozen in a solution comprising DMSO. In some embodiments of any of the methods described herein, the method comprises storing the frozen blood sample at a temperature below 0°C for at least 1 day. In some embodiments of any of the methods described herein, the frozen blood sample is stored at a temperature below -80°C for at least 1 day. In some embodiments of any of the methods described herein, the frozen blood sample is stored at a temperature below -190°C for at least 1 day.

[0037] Some embodiments of any of the methods described herein further include thawing the frozen blood sample and performing an additional immunological test / analysis on the thawed blood sample.

[0038] In some embodiments of any of the methods described herein, the method includes performing low-frequency screening for cancer or infectious disease on subjects identified as having an improved prognosis for cancer or infectious disease.

[0039] In some embodiments of any of the methods described herein, the method includes performing frequent screening for cancer or infectious disease on subjects identified as having a poor prognosis for cancer or infectious disease.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.

[0041] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0042] [Figure 1] 1 is an exemplary image of mononuclear cell blasts. [Diagram 2] 1 is an exemplary image of a large granular lymphocyte. [Diagram 3] 1 is an exemplary image of a lymphocyte. [Figure 4] 1 is an exemplary image of unstimulated lymphocytes. [Diagram 5] 1 is an exemplary image of stimulated lymphocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description Described herein is a method for determining the prognosis of cancer and / or infectious disease in a subject.The method comprises determining the number of viable blast mononuclear cells and / or viable large granular lymphocytes in a blood sample obtained from the subject.

[0044] Mononuclear cell blasts Mononuclear cells refer to blood cells with a single round nucleus. They are called peripheral blood mononuclear cells (PBMCs) when isolated from circulating blood, although other sources exist, such as umbilical cord, spleen, and bone marrow. For example, human PBMCs can be isolated from peripheral blood and identified as any blood cell with a round nucleus. PBMCs can include lymphocytes, monocytes, natural killer cells (NK cells), and dendritic cells. Cell fractions corresponding to red blood cells and granulocytes (neutrophils, basophils, and eosinophils) can be removed from whole blood by density gradient centrifugation (e.g., by using Ficoll, a polysaccharide). A gradient medium with a density of 1.077 g / mL can separate whole blood into two fractions; PBMCs constitute the cell population that remains in the low density fraction (top fraction), while red blood cells and polymorphonuclear leukocytes (PMNs) (including neutrophils, eosinophils, basophils, and mast cells) have a higher density and are found in the bottom fraction. After spinning, the PBMCs collect in a layer called the buffy coat, which constitutes approximately 1% of the total volume of the sample. From this fraction, more specific cell types can be further isolated by purification methods targeting specific cell surface proteins, such as CD4 for helper T cells, CD8 for cytotoxic T cells, CD19 for B cells, and CD14 / CD16 for monocytes, among others. Mononuclear cells are described in more detail in the art, for example, by Kleiveland (“Peripheral blood mononuclear cells,” in The Impact of Food Bioactives on Health, Eds. K. Verhoeckx, P. Cotter, I. Lopez-Exposito, C. Kleiveland, T. Lea, A. Mackie, T. Requena, D. Swiatecka, H. Wichers (Springer), 161-167 (2015)); and Sen et al. (“Perspectives on Systems Modeling of Human Peripheral Blood Mononuclear Cells,” Front Mol. Biosci. 4:96 (2018)).

[0045] Blastification refers to the morphological change of mononuclear cells, such as lymphocytes (e.g., B or T lymphocytes) in culture, into megaloblastoid cells capable of synthesizing DNA and RNA and dividing mitotically. Blastification can be induced by interleukins, mitogens (such as phytohemagglutinin), and by specific antigens. In vivo, blastification of mononuclear cells can be in response to infectious and / or neoplastic conditions, or to transplant rejection. Blastification of lymphocytes by phytohemagglutinin (a nonspecific mitogenic lectin) exhibits the same type of intracellular changes and subsequent development into lymphoblasts that occur in vivo in primed lymphocytes when they are reintroduced to the priming antigen.

[0046] As used herein, blasted mononuclear cells refer to lymphocytes whose cell morphology has been altered. In certain cases, blasted mononuclear cells have larger nuclei compared to non-blasted mononuclear cells. For example, blasted mononuclear cells can have nuclei that are at least about 10% larger than those of non-blasted mononuclear cells (e.g., at least about 20% larger, at least about 30% larger, at least about 40% larger, at least about 50% larger, at least about 60% larger, at least about 70% larger, at least about 80% larger, at least about 90% larger, or at least about 100% larger). Additionally, or alternatively, the nuclear membrane of blasted mononuclear cells can be more irregular compared to those of non-blasted mononuclear cells. An exemplary image of blasted mononuclear cells is shown in FIG. 1. A mononuclear cell blast may be formed by blasting of a lymphocyte (e.g., a B or T lymphocyte), for example, when the lymphocyte is activated by an antigen and increases in volume by nuclear and cytoplasmic growth and synthesis of new mRNA and proteins. The mononuclear cell blast may then begin to divide 2-4 times every 24 hours for 3-5 days, with a single mononuclear cell blast producing approximately 1000 clones of its original naive lymphocyte, each clone sharing the original unique antigen specificity. Finally, the dividing cells may differentiate into effector cells known as plasma cells (in the case of B cells), cytotoxic T cells, and helper T cells. See, for example, Janeway's Immunobiology, 9th edition, Chapter 1, page 23. Mononuclear cell blasts may have a size of about 10 μm to about 20 μm, and compared to myeloblasts, mononuclear cell blasts may have less distinct nucleoli, more condensed chromatin, and / or no cytoplasmic granules.

[0047] An increased number or level (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase) as compared to a reference level (e.g., any of the exemplary reference levels described herein). , at least an 80% increase, at least an 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, at least a 320% increase, at least a 340% increase, at least a 360% increase, at least a 380% increase, or at least at least a 400% increase, or an increase of about 1% to about 400% increase, an increase of about 1% to about 380% increase, an increase of about 1% to about 360% increase, an increase of about 1% to about 340% increase, an increase of about 1% to about 320% increase, an increase of about 1% to about 300% increase, an increase of about 1% to about 280% increase, an increase of about 1% to about 260% increase, an increase of about 1% to about 240% increase, an increase of about 1% to about 220% increase, an increase of about 1% to about 200% increase, an increase of about 1% to about 180% increase, an increase of about 1% to about 160% increase, an increase of about 1% to about 140% increase, an increase of about 1% to about 12 0% increase, about 1% increase to about 100% increase, about 1% increase to about 90% increase, about 1% increase to about 80% increase, about 1% increase to about 70% increase, about 1% increase to about 60% increase, about 1% increase to about 50% increase, about 1% increase to about 40% increase, about 1% increase to about 30% increase, about 1% increase to about 25% increase, about 1% increase to about 20% increase, about 1% increase to about 15% increase, about 1% increase to about 10% increase, about 1% increase to about 5% increase, about 5% increase to about 400% increase, about 5% increase to about 380% increase, about 5% increase to about 360% increase,5% increase to 340% increase, 5% increase to 320% increase, 5% increase to 300% increase, 5% increase to 280% increase, 5% increase to 260% increase, 5% increase to 240% increase, 5% increase to 220% increase, 5% increase to 200% increase, 5% increase to 180% increase, 5% increase to 160% increase, 5% increase to 140% increase, 5% increase to 120% increase, 5% increase to 100% increase, 5% increase to 90% increase, 5% increase to 80% increase, 5% increase to 70 % increase, about 5% increase to about 60% increase, about 5% increase to about 50% increase, about 5% increase to about 40% increase, about 5% increase to about 30% increase, about 5% increase to about 25% increase, about 5% increase to about 20% increase, about 5% increase to about 15% increase, about 5% increase to about 10% increase, about 10% increase to about 400% increase, about 10% increase to about 380% increase, about 10% increase to about 360% increase, about 10% increase to about 340% increase, about 10% increase to about 320% increase, about 10% increase to about 300% increase, about 10% increase to about 280% increase, about 10% increase ~ about 260% increase, about 10% increase ~ about 240% increase, about 10% increase ~ about 220% increase, about 10% increase ~ about 200% increase, about 10% increase ~ about 180% increase, about 10% increase ~ about 160% increase, about 10% increase ~ about 140% increase, about 10% increase ~ about 120% increase, about 10% increase ~ about 100% increase, about 10% increase ~ about 90% increase, about 10% increase ~ about 80% increase, about 10% increase ~ about 70% increase, about 10% increase ~ about 60% increase, about 10% increase ~ about 50% increase, about 10% increase ~ about 40% increase, about 10 % increase to about 30% increase, about 10% increase to about 25% increase, about 10% increase to about 20% increase, about 10% increase to about 15% increase, about 15% increase to about 400% increase, about 15% increase to about 380% increase, about 15% increase to about 360% increase, about 15% increase to about 340% increase, about 15% increase to about 320% increase, about 15% increase to about 300% increase, about 15% increase to about 280% increase, about 15% increase to about 260% increase, about 15% increase to about 240% increase, about 15% increase to about 220% increase, about 15% increase to about 200% increase,15% increase to 180% increase, 15% increase to 160% increase, 15% increase to 140% increase, 15% increase to 120% increase, 15% increase to 100% increase, 15% increase to 90% increase, 15% increase to 80% increase, 15% increase to 70% increase, 15% increase to 60% increase, 15% increase to 50% increase, 15% increase to 40% increase, 15% increase to 30% increase, 15% increase to 25% increase, 15% increase to 20% increase, 20% increase to 400% increase, 20% increase to 400% increase % increase to about 380% increase, about 20% increase to about 360% increase, about 20% increase to about 340% increase, about 20% increase to about 320% increase, about 20% increase to about 300% increase, about 20% increase to about 280% increase, about 20% increase to about 260% increase, about 20% increase to about 240% increase, about 20% increase to about 220% increase, about 20% increase to about 200% increase, about 20% increase to about 180% increase, about 20% increase to about 160% increase, about 20% increase to about 140% increase, about 20% increase to about 120% increase, about 20% increase to about 100% increase, about 20% increase to about 90% increase, about 20% increase to about 80% increase, about 20% increase to about 70% increase, about 20% increase to about 60% increase, about 20% increase to about 50% increase, about 20% increase to about 40% increase, about 20% increase to about 30% increase, about 20% increase to about 25% increase, about 25% increase to about 400% increase, about 25% increase to about 380% increase, about 25% increase to about 360% increase, about 25% increase to about 340% increase, about 25% increase to about 320% increase, about 25% increase to about 300% increase, about 25% increase to about 280% increase , about 25% increase to about 260% increase, about 25% increase to about 240% increase, about 25% increase to about 220% increase, about 25% increase to about 200% increase, about 25% increase to about 180% increase, about 25% increase to about 160% increase, about 25% increase to about 140% increase, about 25% increase to about 120% increase, about 25% increase to about 100% increase, about 25% increase to about 90% increase, about 25% increase to about 80% increase, about 25% increase to about 70% increase, about 25% increase to about 60% increase, about 25% increase to about 50% increase, about 25% increase to about 40% increase,25% increase ~ 30% increase, 30% increase ~ 400% increase, 30% increase ~ 380% increase, 30% increase ~ 360% increase, 30% increase ~ 340% increase, 30% increase ~ 320% increase, 30% increase ~ 300% increase, 30% increase ~ 280% increase, 30% increase ~ 260% increase, 30% increase ~ 240% increase, 30% increase ~ 220% increase, 30% increase ~ 200% increase, 30% increase ~ 180% increase, 30% increase ~ 160% increase, 30% increase ~ 1 40% increase, about 30% increase to about 120% increase, about 30% increase to about 100% increase, about 30% increase to about 90% increase, about 30% increase to about 80% increase, about 30% increase to about 70% increase, about 30% increase to about 60% increase, about 30% increase to about 50% increase, about 30% increase to about 40% increase, about 40% increase to about 400% increase, about 40% increase to about 380% increase, about 40% increase to about 360% increase, about 40% increase to about 340% increase, about 40% increase to about 320% increase, about 40% increase to about 300% increase, about 40% increase to about 280% increase, 40% increase to 260% increase, 40% increase to 240% increase, 40% increase to 220% increase, 40% increase to 200% increase, 40% increase to 180% increase, 40% increase to 160% increase, 40% increase to 140% increase, 40% increase to 120% increase, 40% increase to 100% increase, 40% increase to 90% increase, 40% increase to 80% increase, 40% increase to 70% increase, 40% increase to 60% increase, 40% increase to 50% increase, 50% increase ~400% increase, ~50% increase~380% increase, ~50% increase~360% increase, ~50% increase~340% increase, ~50% increase~320% increase, ~50% increase~300% increase, ~50% increase~280% increase, ~50% increase~260% increase, ~50% increase~240% increase, ~50% increase~220% increase, ~50% increase~200% increase, ~50% increase~180% increase, ~50% increase~160% increase, ~50% increase~140% increase, ~50% increase~120% increase,About 50% increase to about 100% increase, about 50% increase to about 90% increase, about 50% increase to about 80% increase, about 50% increase to about 70% increase, about 50% increase to about 60% increase, about 60% increase to about 400% increase, about 60% increase to about 380% increase, about 60% increase to about 360% increase, about 60% increase to about 340% increase, about 60% increase to about 320% increase, about 60% increase to about 300% increase, about 60% increase to about 280% increase, about 60% increase to about 260% increase, about 60% increase to about 240% increase, about 60% increase to about 220% increase increase, about 60% increase to about 200% increase, about 60% increase to about 180% increase, about 60% increase to about 160% increase, about 60% increase to about 140% increase, about 60% increase to about 120% increase, about 60% increase to about 100% increase, about 60% increase to about 90% increase, about 60% increase to about 80% increase, about 60% increase to about 70% increase, about 70% increase to about 400% increase, about 70% increase to about 380% increase, about 70% increase to about 360% increase, about 70% increase to about 340% increase, about 70% increase to about 320% increase, about 70% increase to about 3 00% increase, about 70% increase to about 280% increase, about 70% increase to about 260% increase, about 70% increase to about 240% increase, about 70% increase to about 220% increase, about 70% increase to about 200% increase, about 70% increase to about 180% increase, about 70% increase to about 160% increase, about 70% increase to about 140% increase, about 70% increase to about 120% increase, about 70% increase to about 100% increase, about 70% increase to about 90% increase, about 70% increase to about 80% increase, about 80% increase to about 400% increase, about 80% increase to about 380% increase, about 80% Increase ~ about 360% increase, about 80% increase ~ about 340% increase, about 80% increase ~ about 320% increase, about 80% increase ~ about 300% increase, about 80% increase ~ about 280% increase, about 80% increase ~ about 260% increase, about 80% increase ~ about 240% increase, about 80% increase ~ about 220% increase, about 80% increase ~ about 200% increase, about 80% increase ~ about 180% increase, about 80% increase ~ about 160% increase, about 80% increase ~ about 140% increase, about 80% increase ~ about 120% increase, about 80% increase ~ about 100% increase, about 80% increase ~ about 90% increase,90% increase~400% increase, 90% increase~380% increase, 90% increase~360% increase, 90% increase~340% increase, 90% increase~320% increase, 90% increase~300% increase, 90% increase~280% increase, 90% increase~260% increase, 90% increase~240% increase, 90% increase~220% increase, 90% increase~200% increase, 90% increase~180% increase, 90% increase~160% increase, 90% increase~140% increase, 90% increase~1 20% increase, 90% increase~100% increase, 100% increase~400% increase, 100% increase~380% increase, 100% increase~360% increase, 100% increase~340% increase, 100% increase~320% increase, 100% increase~300% increase, 100% increase~280% increase, 100% increase~260% increase, 100% increase~240% increase, 100% increase~220% increase, 100% increase~200% increase, 100% increase~180% increase, 100% increase ~160% increase, ~100% increase~140% increase, ~100% increase~120% increase, ~120% increase~400% increase, ~120% increase~380% increase, ~120% increase~360% increase, ~120% increase~340% increase, ~120% increase~320% increase, ~120% increase~300% increase, ~120% increase~280% increase, ~120% increase~260% increase, ~120% increase~240% increase, ~120% increase~220% increase, ~120% increase~200% increase, ~120 % increase ~ approx. 180% increase, approx. 120% increase ~ approx. 160% increase, approx. 120% increase ~ approx. 140% increase, approx. 140% increase ~ approx. 400% increase, approx. 140% increase ~ approx. 380% increase, approx. 140% increase ~ approx. 360% increase, approx. 140% increase ~ approx. 340% increase, approx. 140% increase ~ approx. 320% increase, approx. 140% increase ~ approx. 300% increase, approx. 140% increase ~ approx. 280% increase, approx. 140% increase ~ approx. 260% increase, approx. 140% increase ~ approx. 240% increase, approx. 140% increase ~ approx. 220% increase, approx. 140% increase ~ approx. 200% increase,140% increase ~ 180% increase, 140% increase ~ 160% increase, 160% increase ~ 400% increase, 160% increase ~ 380% increase, 160% increase ~ 360% increase, 160% increase ~ 340% increase, 160% increase ~ 320% increase, 160% increase ~ 300% increase, 160% increase ~ 280% increase, 160% increase ~ 260% increase, 160% increase ~ 240% increase, 160% increase ~ about 220% increase, about 160% increase ~ about 200% increase, about 160% increase ~ about 180% increase, about 180% increase ~ about 400% increase, about 180% increase ~ about 380% increase, about 180% increase ~ about 360% increase, about 180% increase ~ about 340% increase, about 180% increase ~ about 320% increase, about 180% increase ~ about 300% increase, about 180% increase ~ about 280% increase, about 180% increase ~ about 260% increase, about 180% increase ~, Approximately 240% increase, approximately 180% increase to approximately 220% increase, approximately 180% increase to approximately 200% increase, approximately 200% increase to approximately 400% increase, approximately 200% increase to approximately 380% increase, approximately 200% increase to approximately 360% increase, approximately 200% increase to approximately 340% increase, approximately 200% increase to approximately 320% increase, approximately 200% increase to approximately 300% increase, approximately 200% increase to approximately 280% increase, approximately 200% increase to approximately 260% increase, approximately 200% increase to approximately 240% increase, approximately 200% increase to approximately 220% increase, approximately 220% increase to approximately 400% increase, approximately 220% increase~approx. 380% increase, increase of about 220%~approx. 360% increase, increase of about 220%~approx. 340% increase, increase of about 220%~approx. 320% increase, increase of about 220%~approx. 300% increase, increase of about 220%~approx. 280% increase, increase of about 220%~approx. 260% increase, increase of about 220%~approx. 240% increase, increase of about 240%~approx. 400% increase, increase of about 240%~approx. 380% increase, increase of about 240%~approx. 360% increase, increase of about 240%~approx. 340% increase, increase of about 240%~approx. 320% increase, increase of about 240%~approx. 300% increase, increase of about 240 % increase~approx. 280% increase, approx. 240% increase~approx. 260% increase, approx. 260% increase~approx. 400% increase, approx. 260% increase~approx. 380% increase, approx. 260% increase~approx. 360% increase, approx. 260% increase~approx. 340% increase, approx. 260% increase~approx. 320% increase, approx. 260% increase~approx. 300% increase, approx. 260% increase~approx. 280% increase, approx. 280% increase~approx. 400% increase, approx. 280% increase~approx. 380% increase, approx. 280% increase~approx. 360% increase, approx. 280% increase~approx. 340% increase, approx. 280% increase~approx. 320% increase, approx. 80% increase~approx. 300% increase, approx. 300% increase~approx. 400% increase, approx. 300% increase~approx. 380% increase, approx. 300% increase~approx. 360% increase, approx. 300% increase~approx. 340% increase, approx. 300% increase~approx. 320% increase, approx. 320% increase~approx. 400% increase, approx. 320% increase~approx. 380% increase, approx. 320% increase~approx. 360% increase, approx. 320% increase~approx. 340% increase, approx. 340% increase~approx. 400% increase, approx. 340% increase~approx. 380% increase, approx. 340% increase~approx. 360% increase, approx. 360% increase~approx. 400% increase,A subject having an increased number of viable mononuclear cell blasts (about a 360% increase to about a 380% increase, or about a 380% increase to about a 400% increase) can be identified as having an improved prognosis for cancer and / or infectious disease. For example, a subject having an increased number of viable mononuclear cell blasts compared to a reference level (e.g., any of the exemplary reference levels described herein) can be identified as having an improved prognosis for cancer or infectious disease.

[0048] Instead, a decreased number or level (e.g., at least a 1% decrease, at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 80% decrease, at least a 90% decrease, at least a 100% decrease, at least a 150% decrease, at least a 200% decrease, at least a 300% decrease, at least a 350% decrease, at least a 400% decrease, at least a 450% decrease, at least a 500% decrease, at least a 550% decrease, at least a 600% decrease, at least a 650% decrease, at least a 700% decrease, at least a 100% decrease, at least a 15 ... at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, or about a 1% to about a 99% reduction, about a 1% to about a 95% reduction, about a 1% to about a 95% reduction, about a 1% to about a 90% reduction, about a 1% to about a 85% reduction, about a 1% to about a 80% reduction, about a 1% to about a 75% reduction, about a 1% to about a 70% reduction, about a 1% to about a 65% reduction, about a 1% to about a 60% reduction, about a 1% to about a 55% reduction, about a 1% Decrease to about 50% decrease, Decrease of about 1% to about 45% decrease, Decrease of about 1% to about 40% decrease, Decrease of about 1% to about 35% decrease, Decrease of about 1% to about 30% decrease, Decrease of about 1% to about 25% decrease, Decrease of about 1% to about 20% decrease, Decrease of about 1% to about 15% decrease, Decrease of about 1% to about 10% decrease, Decrease of about 1% to about 5% decrease, Decrease of about 5% to about 99% decrease, Decrease of about 5% to about 95% decrease, Decrease of about 5% to about 95% decrease, Decrease of about 5% to about 90% decrease, Decrease of about 5% to about 85% decrease, Decrease of about 5% to about 80% decrease, Decrease of about 5% to about 75% reduction of about 5% to about 70% reduction, reduction of about 5% to about 65% reduction, reduction of about 5% to about 60% reduction, reduction of about 5% to about 55% reduction, reduction of about 5% to about 50% reduction, reduction of about 5% to about 45% reduction, reduction of about 5% to about 40% reduction, reduction of about 5% to about 35% reduction, reduction of about 5% to about 30% reduction, reduction of about 5% to about 25% reduction, reduction of about 5% to about 20% reduction, reduction of about 5% to about 15% reduction, reduction of about 5% to about 10% reduction, reduction of about 10% to about 99% reduction, reduction of about 10% to about 95% reduction, reduction of about 10% to about 95% reduction,About 10% decrease to about 90% decrease, About 10% decrease to about 85% decrease, About 10% decrease to about 80% decrease, About 10% decrease to about 75% decrease, About 10% decrease to about 70% decrease, About 10% decrease to about 65% decrease, About 10% decrease to about 60% decrease, About 10% decrease to about 55% decrease, About 10% decrease to about 50% decrease, About 10% decrease to about 45% decrease, About 10% decrease to about 40% decrease, About 10% decrease to about 35% decrease, About 10% decrease to about 30% decrease, About 10% decrease to about 25% decrease, About 10% decrease to about 20% decrease, About 10% decrease to About 15% reduction, About 15% reduction to about 99% reduction, About 15% reduction to about 95% reduction, About 15% reduction to about 95% reduction, About 15% reduction to about 90% reduction, About 15% reduction to about 85% reduction, About 15% reduction to about 80% reduction, About 15% reduction to about 75% reduction, About 15% reduction to about 70% reduction, About 15% reduction to about 65% reduction, About 15% reduction to about 60% reduction, About 15% reduction to about 55% reduction, About 15% reduction to about 50% reduction, About 15% reduction to about 45% reduction, About 15% reduction to about 40% reduction, About 15% reduction to about 35% reduction, About 15% decrease to about 30% decrease, About 15% decrease to about 25% decrease, About 15% decrease to about 20% decrease, About 20% decrease to about 99% decrease, About 20% decrease to about 95% decrease, About 20% decrease to about 95% decrease, About 20% decrease to about 90% decrease, About 20% decrease to about 85% decrease, About 20% decrease to about 80% decrease, About 20% decrease to about 75% decrease, About 20% decrease to about 70% decrease, About 20% decrease to about 65% decrease, About 20% decrease to about 60% decrease, About 20% decrease to about 55% decrease, About 20% decrease to about 50% decrease, About 20% decrease to About 45% reduction, About 20% reduction to about 40% reduction, About 20% reduction to about 35% reduction, About 20% reduction to about 30% reduction, About 20% reduction to about 25% reduction, About 25% reduction to about 99% reduction, About 25% reduction to about 95% reduction, About 25% reduction to about 95% reduction, About 25% reduction to about 90% reduction, About 25% reduction to about 85% reduction, About 25% reduction to about 80% reduction, About 25% reduction to about 75% reduction, About 25% reduction to about 70% reduction, About 25% reduction to about 65% reduction, About 25% reduction to about 60% reduction, About 25% reduction to about 55% reduction,About 25% decrease to about 50% decrease, About 25% decrease to about 45% decrease, About 25% decrease to about 40% decrease, About 25% decrease to about 35% decrease, About 25% decrease to about 30% decrease, About 30% decrease to about 99% decrease, About 30% decrease to about 95% decrease, About 30% decrease to about 95% decrease, About 30% decrease to about 90% decrease, About 30% decrease to about 85% decrease, About 30% decrease to about 80% decrease, About 30% decrease to about 75% decrease, About 30% decrease to about 70% decrease, About 30% decrease to about 65% decrease, About 30% decrease to about 60% decrease, About 30% decrease to About 55% reduction, About 30% reduction to about 50% reduction, About 30% reduction to about 45% reduction, About 30% reduction to about 40% reduction, About 30% reduction to about 35% reduction, About 35% reduction to about 99% reduction, About 35% reduction to about 95% reduction, About 35% reduction to about 95% reduction, About 35% reduction to about 90% reduction, About 35% reduction to about 85% reduction, About 35% reduction to about 80% reduction, About 35% reduction to about 75% reduction, About 35% reduction to about 70% reduction, About 35% reduction to about 65% reduction, About 35% reduction to about 60% reduction, About 35% reduction to about 55% reduction, 35% to 50% reduction, 35% to 45% reduction, 35% to 40% reduction, 40% to 99% reduction, 40% to 95% reduction, 40% to 95% reduction, 40% to 90% reduction, 40% to 85% reduction, 40% to 80% reduction, 40% to 75% reduction, 40% to 70% reduction, 40% to 65% reduction, 40% to 60% reduction, 40% to 55% reduction, 40% to 50% reduction, 40% to About 45% reduction, About 45% reduction to about 99% reduction, About 45% reduction to about 95% reduction, About 45% reduction to about 95% reduction, About 45% reduction to about 90% reduction, About 45% reduction to about 85% reduction, About 45% reduction to about 80% reduction, About 45% reduction to about 75% reduction, About 45% reduction to about 70% reduction, About 45% reduction to about 65% reduction, About 45% reduction to about 60% reduction, About 45% reduction to about 55% reduction, About 45% reduction to about 50% reduction, About 50% reduction to about 99% reduction, About 50% reduction to about 95% reduction, About 50% reduction to about 95% reduction,About 50% reduction to about 90% reduction, About 50% reduction to about 85% reduction, About 50% reduction to about 80% reduction, About 50% reduction to about 75% reduction, About 50% reduction to about 70% reduction, About 50% reduction to about 65% reduction, About 50% reduction to about 60% reduction, About 50% reduction to about 55% reduction, About 55% reduction to about 99% reduction, About 55% reduction to about 95% reduction, About 55% reduction to about 95% reduction, About 55% reduction to about 90% reduction, About 55% reduction to about 85% reduction, About 55% reduction to about 80% reduction, About 55% reduction to about 75% reduction, About 55% reduction to About 70% reduction, About 55% reduction to about 65% reduction, About 55% reduction to about 60% reduction, About 60% reduction to about 99% reduction, About 60% reduction to about 95% reduction, About 60% reduction to about 95% reduction, About 60% reduction to about 90% reduction, About 60% reduction to about 85% reduction, About 60% reduction to about 80% reduction, About 60% reduction to about 75% reduction, About 60% reduction to about 70% reduction, About 60% reduction to about 65% reduction, About 65% reduction to about 99% reduction, About 65% reduction to about 95% reduction, About 65% reduction to about 95% reduction, About 65% reduction to about 90% reduction, About 65% reduction to about 85% reduction, About 65% reduction to about 80% reduction, About 65% reduction to about 75% reduction, About 65% reduction to about 70% reduction, About 70% reduction to about 99% reduction, About 70% reduction to about 95% reduction, About 70% reduction to about 95% reduction, About 70% reduction to about 90% reduction, About 70% reduction to about 85% reduction, About 70% reduction to about 80% reduction, About 70% reduction to about 75% reduction, About 75% reduction to about 99% reduction, About 75% reduction to about 95% reduction, About 75% reduction to about 95% reduction, About 75% reduction to about 90% reduction, About 75% reduction to about 95% reduction About 85% reduction, About 75% reduction to about 80% reduction, About 80% reduction to about 99% reduction, About 80% reduction to about 95% reduction, About 80% reduction to about 95% reduction, About 80% reduction to about 90% reduction, About 80% reduction to about 85% reduction, About 85% reduction to about 99% reduction, About 85% reduction to about 95% reduction, About 85% reduction to about 95% reduction, About 85% reduction to about 90% reduction, About 90% reduction to about 99% reduction, About 90% reduction to about 95% reduction, About 90% reduction to about 95% reduction, About 95% reduction to about 99% reduction, About 95% reduction to about 95% reduction,Subjects having a low or no viable mononuclear cell blast count (or about a 95% reduction to about a 99% reduction) can be identified as having a poor prognosis for cancer and / or infectious diseases.

[0049] In certain instances, the reference level is about the 70th percentile, about the 71st percentile, about the 72nd percentile, about the 73rd percentile, about the 74th percentile, about the 75th percentile, about the 76th percentile, about the 77th percentile, about the 78th percentile, about the 79th percentile, about the 80th percentile, about the 81st percentile, about the 82nd percentile, about the 83rd percentile, about the 84th percentile, about the 85th percentile, about the 86th percentile, about the 87th percentile, about the 88th percentile, about the 89th percentile, about the 90th percentile, about the 91st percentile, about the 92nd percentile, about the 93rd percentile, about the 94th percentile, about the 95th percentile, about the 96th percentile, about the 97th percentile, about the 98th percentile, or about the 99th percentile) of the median level of the number or level of viable mononuclear cell blasts in a healthy patient population. A healthy patient population can be a patient population that is not currently diagnosed with cancer and / or an infectious disease or has not been diagnosed with cancer and / or an infectious disease within the past 5 years (e.g., within the past 4 years, within 3 years, within 2 years, within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, or within 1 week).

[0050] Large granular lymphocytes Large granular lymphocytes (LGLs) are lymphoid cells characterized by either a T cell or natural killer (NK) phenotype that are physiologically involved in innate immunity and immune surveillance. Morphologically, LGLs are characterized by round or kidney-shaped nuclei more than twice the diameter of red blood cells, mature chromatin, abundant cytoplasm, and prominent azurophilic cytoplasmic granules. Typically, LGLs constitute 10%-15% of blood mononuclear cells and express surface CD3 + (T cells) or surface CD3 -Large granular lymphocytes can be either NK cells or T lymphocytes. Most normal large granular lymphocytes in peripheral blood are NK cells, but some are T lymphocytes. Large granular lymphocytes have been described in more detail in the art, for example, by Fattizzo et al. ("Large Granular Lymphocyte Expansion in Myeloid Diseases and Bone Marrow Failure Syndromes: Whoever Seeks Finds," Front. Oncol. 11:748610 (2021)); Vivier et al. ("Innate or adaptive immunity? The example of natural killer cells," Science 331(6013):44-49 (2011)); and Oshimi ("Clinical Features, Pathogenesis, and Treatment of Large Granular Lymphocyte Leukemias," Intern. Med. 56:1759-1769 (2017)). An exemplary image of a large granular lymphocyte is shown in FIG. 2. Exemplary images of lymphocytes, unstimulated lymphocytes, and stimulated lymphocytes are shown in Figures 3-5, respectively.

[0051] In some embodiments, the large granular lymphocytes are NK cells. NK cells provide a rapid response to virus-infected cells and other intracellular pathogens, acting as early as 3 days after infection, and also respond to tumor formation. Typically, immune cells detect major histocompatibility complexes (MHC) displayed on the infected cell surface, triggering cytokine release and causing the death of the infected cell by lysis or apoptosis. However, NK cells are unique because they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing the immune response to be much faster. They were named "natural killers" due to the notion that they do not require activation to kill cells that lack the "self" marker of MHC class I. This role is particularly important because harmful cells that lack the MHC I marker cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.

[0052] As described herein, an increased number or level (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 10 ... Subjects having at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, at least a 320% increase, at least a 340% increase, at least a 360% increase, at least a 380% increase, or at least a 400% increase, or between about a 1% increase and about a 400% increase (or any sub-range of this range described herein)) of viable large granular lymphocytes may be identified as having an improved prognosis of one or more cancers and / or infectious diseases.

[0053] In other examples, subjects with a decreased number or level (e.g., at least a 1% decrease, at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, at least a 95% decrease, or at least a 99% decrease, or between about a 1% decrease and about a 99% decrease (or any sub-range of this range described herein)) of viable large granular lymphocytes compared to a reference level (e.g., any of the exemplary reference levels described herein).

[0054] In certain instances, the reference level is about the 70th percentile, about the 71st percentile, about the 72nd percentile, about the 73rd percentile, about the 74th percentile, about the 75th percentile, about the 76th percentile, about the 77th percentile, about the 78th percentile, about the 79th percentile, about the 80th percentile, about the 81st percentile, about the 82nd percentile, about the 83rd percentile, about the 84th percentile, about the 85th percentile, about the 86th percentile, about the 87th percentile, about the 88th percentile, about the 89th percentile, about the 90th percentile, about the 91st percentile, about the 92nd percentile, about the 93rd percentile, about the 94th percentile, about the 95th percentile, about the 96th percentile, about the 97th percentile, about the 98th percentile, or about the 99th percentile) of the median level of the number or level of viable large granular lymphocytes in a healthy patient population. A healthy patient population can be a patient population that is not currently diagnosed with cancer and / or an infectious disease or has not been diagnosed with cancer and / or an infectious disease within the past 5 years (e.g., within the past 4 years, within 3 years, within 2 years, within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, or within 1 week).

[0055] Sample preparation The present disclosure provides a method for assessing the prognosis of a subject's cancer and / or infectious disease by analyzing a blood sample from the subject. Described herein are methods for collecting and preparing blood samples for use in the methods of the present invention.

[0056] Collection of blood samples Blood samples can be collected from subjects by standard phlebotomy methods known in the art (see, for example, WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy. Geneva: World Health Organization; 2010. 2, Best practices in phlebotomy). In certain cases, blood samples can be collected with anticoagulants such as heparin, ethylenediaminetetraacetic acid (EDTA), citrate, acid citrate dextrose (ACD), or citrate phosphate dextrose (CPD). A blood sample for use in the methods of the present invention can be a venous blood sample. A venous blood sample can be collected from a subject by venipuncture. Additionally, or alternatively, a blood sample for use in the methods of the present invention can be a capillary blood sample. A capillary blood sample can be a fingerstick blood sample (e.g., collected from a subject by a fingerstick technique, such as by pricking a finger) or a heelstick blood sample (e.g., collected from a subject by a heelstick technique, such as by pricking a heel).

[0057] While it is generally preferred to prepare cells for analysis immediately after collection of the blood sample, in some cases (e.g., transporting specimens from a remote location), it may be necessary to store the blood sample for a short period of time prior to further processing (e.g., prior to isolation and analysis of PBMCs). For example, blood samples may be stored for at least 12 hours (e.g., at least 12-18 hours, 18-24 hours, 1-2 days, 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-3 months, 1-4 months, 1-5 months, 1-6 months, or more (e.g., at least 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more). Several commercially available reagents can be used to treat blood samples for storage.For example, when used in the method of the present invention, blood samples can be stored using TRANSFIX (CYTOMARK) and / or CYTO-CHEX (STRECK).In addition, or instead, blood samples can also be stored in dimethyl sulfoxide (DMSO).

[0058] In some cases, the blood sample may be stored at room temperature. In some cases, the blood sample may be stored at a temperature of about 0°C to about 21°C (e.g., about 1°C to about 21°C, about 4°C to about 21°C, about 10°C to about 21°C, about 18°C ​​to about 21°C, about 0°C to about 18°C, about 1°C to about 18°C, about 4°C to about 18°C, about 10°C to about 18°C, about 0°C to about 10°C, about 1°C to about 10°C, or about 4°C to about 10°C (e.g., about 0°C, 1°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, or 21°C)). In certain cases, blood samples may be frozen and stored at temperatures below 0° C. (e.g., below -1° C., below -10° C., below -20° C., below -30° C., below -40° C., below -50° C., below -60° C., below -70° C., below -80° C., below -90° C., below -100° C., below -110° C., below -120° C., below -130° C., below -140° C., below -150° C., below -160° C., below -170° C., below -180° C., below -190° C., or below -200° C.). Frozen blood samples may be thawed prior to further processing (e.g., prior to isolating PBMCs from such blood samples and / or preparing microscope slides as described herein). Each assay in which blood samples have been stored (e.g., frozen and stored) may be validated on the stored specimen so that assay data from a fresh specimen can be compared to assay data from the same specimen after storage to ensure similar, if not identical, data are obtained.

[0059] Cell isolation Cells can be isolated from blood samples using methods known in the art. See, for example, Dagur and McCoy JP Jr., "Collection, Storage, and Preparation of Human Blood Cells," Curr. Protoc. Cytom. 73:5.1.1-5.1.16 (2015).

[0060] In certain cases, PBMCs (e.g., lymphocytes, NK cells, etc.) can be isolated from blood samples by removing red blood cells from the sample, such as by lysis. Lysis is more rapid than gradient separation and generally leaves the remaining white blood cell population relatively undisturbed. Furthermore, the yield of mononuclear cells from blood by lysis of red blood cells is much higher than that from density gradient separation. This technique, in which red blood cells are lysed by osmotic shock (e.g., cell membrane lysis with ammonium chloride), can be used on unstained blood or blood that has already been incubated with monoclonal antibodies. Generally, this method does not affect the staining patterns observed for most lymphoid markers. The viability of white blood cells subjected to this treatment is good. Red blood cells can be lysed using homemade RBC lysis buffer and commercial RBC lysis buffer without fixative, such as ACK LYSIS BUFFER (Quality Biological), OPTILYSE (Beckman Coulter), PHARMLYSE (BD Bioscience), IOTEST3 (Beckman Coulter) HI-YIELD LYSE (Life Technologies), or EASY-LYSE ERYTHROCYTE LYSIS SOLUTION (Dako). Additionally, or alternatively, red blood cells can be lysed by using a lysis reagent containing a fixative. For example, commercial reagents such as FACSLYSE (BD Bioscience) or RBC LYSIS / FIXATION SOLUTION (BioLegend) can be used instead of ammonium chloride to lyse red blood cells. However, since FACSLYSE contains a fixative, staining for cell surface markers on PBMCs must be performed before lysing red blood cells with this reagent. For example, whole blood samples can be stained with antibodies as needed, and then red blood cells can be lysed according to the manufacturer's instructions. Additionally or alternatively, OPTILYSE (Beckman Coulter) can be used to lyse red blood cells without subsequent washing of the PBMCs.

[0061] In certain cases, PBMCs (e.g., lymphocytes, NK cells, etc.) may be isolated from blood samples by density gradient centrifugation (see, e.g., Boyum, "Isolation of mononuclear cells and granulocytes from human blood," Scand. J. Clin. Lab Invest. Suppl. 21:77-89 (1968); and Boyum, "Separation of lymphocytes, lymphocyte subgroups and monocytes: A review," Lymphology 10:71-76 (1977)). Isolation of PBMCs by density gradient centrifugation may be useful when purification of a cell population is required rather than simple removal of red blood cell contaminants. Although density gradient separation techniques do not yield as many PBMCs as simple lysis methods, this method has other distinct advantages. The primary advantage of this method is the removal of most granulocytes from the sample. An additional advantage is the removal of non-viable cells from the sample. There are modifications of this method to enrich for unlabeled cells of interest, for example, by using ROSETTESEP TETRAMERIC ANTIBODY COMPLEXES (a cocktail of bispecific antibodies to preferentially crosslink undesired cells to RBCs; Stem Cell Technologies), which crosslinks and pellets undesired cells together with RBCs to remove them. This leaves only unlabeled cells of interest for studies such as those described herein. A medium with a density of 1.077 g / mL can be used for the isolation of PBMCs from blood samples by density gradient centrifugation. For example, 1.077 g / mL Ficoll-Hypaque (GE Healthcare) or Histopaque-1077 (Sigma) or Lymphoprep (STEMCELL Technologies) can be used for the isolation of PBMCs from blood samples by density gradient centrifugation.

[0062] Density gradient separation methods for isolating PBMCs are known in the art (see, e.g., Dagur and McCoy JP Jr., "Collection, Storage, and Preparation of Human Blood Cells," Curr. Protoc. Cytom. 73:5.1.1-5.1.16 (2015)). In certain cases, anticoagulated blood can be diluted with an equal volume of PBS and layered onto the Ficoll-Hypaque solution. Layering can be performed by gently pipetting the diluted blood down the side of a Ficoll-Hypaque-containing tube. After centrifugation (e.g., 400×g, 22° C. for 30 minutes, no brake), PBMCs can be collected from the interface between the plasma (top layer) and the Ficoll-Hypaque (bottom layer).

[0063] In certain cases, PBMCs isolated by the methods described above can be enriched for lymphocytes by depleting monocyte-like cells from the mononuclear cell preparation, for example by allowing the monocyte-like cells to adhere to plastic tissue culture flasks.

[0064] Cell viability assay The blood sample and / or cells isolated therefrom (e.g., PBMCs) can be subjected to a dye exclusion test, in which the blood sample and / or cell suspension are contacted with a cell viability dye to determine the number of viable cells. The dye exclusion test is based on the principle that live cells have an intact cell membrane that excludes certain dyes (e.g., cell viability dyes, such as trypan blue, eosin, propidium, etc.), whereas dead cells do not have an intact cell membrane and therefore cannot exclude these dyes. Thus, in this test, the blood sample and / or cell suspension are mixed with a cell viability dye and then visually inspected, e.g., under a microscope, to determine whether the cells take up or exclude the dye. For example, a dye exclusion test can be performed using trypan blue, where viable cells have a clear cytoplasm because the intact cell membrane of live cells excludes the dye, whereas nonviable cells cannot exclude the dye and appear to have a blue cytoplasm when inspected under a microscope.

[0065] Incubation with antigen In some cases, the blood sample and / or cells isolated therefrom (e.g., PBMCs) are incubated with an antigen before further processing. For example, the blood sample and / or cells isolated therefrom (e.g., PBMCs) can be incubated with an antigen specific to an infectious disease, such as COVID-19 (i.e., SARS-CoV2 infection), influenza, cold, human immunodeficiency virus (HIV) infection, herpes virus infection, hepatitits (e.g., hepatitis B virus infection, hepatitis C virus infection), norovirus infection, rotavirus infection, rabies virus infection, measles, mumps, rubella virus infection, chickenpox, whooping cough, West Nile virus infection, meningitis, poliovirus infection, Zika virus infection, cytomegalovirus infection, or enterovirus infection (e.g., coxsackievirus infection, echovirus infection). In certain cases, the infectious disease specific antigen is a vaccine that is routinely used to prevent infectious diseases. For example, a blood sample and / or PBMCs isolated therefrom may be incubated with a COVID-19 vaccine.

[0066] In certain cases, the blood sample and / or cells isolated therefrom (e.g., PBMCs) are incubated for at least about 30 minutes (e.g., at least about 1 hour, at least about 2 hours, about at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days, or from about 1 hour to about 7 days, from about 1 hour to about 6 days, from about 1 hour to about 5 days, from about 1 hour to about 4 days, from about 1 hour to about 3 days, from about 1 hour to about 1 hour, or from about 1 hour to about 1 hour. ~ about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 60 hours, about 2 hours to about 48 hours, about 2 hours to about 36 hours, about 2 hours to about 24 hours, about 2 hours to about 18 hours, about 2 hours to about 12 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours, about 2 hours to about 4 hours, about 2 hours to about 3 hours, about 3 hours to about 7 days, about 3 hours to about 6 days, about 3 hours to about 5 days, about 3 hours to about 4 days, about 3 hours to about 3 days, about 3 hours to about 60 hours, about 3 hours to about 48 hours, about 3 hours to about 36 hours, about 3 hours to about 24 hours, about 3 hours to about 18 hours, about 3 hours to about 12 hours, about 3 hours to about 8 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, about 3 hours to about 4 hours, about 4 hours to about 7 days, about 4 hours to about 6 days, about 4 hours to about 5 days, about 4 hours to about 4 days, about 4 hours to about 3 days, about 4 hours to about 60 hours, about 4 hours to about 48 hours, about 4 hours to about 36 hours, about 4 hours to about 24 hours, about 4 hours to about 18 hours, about 4 hours to about 12 hours, about 4 hours to about 8 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours, about 5 hours to about 7 days, about 5 hours to about 6 days, about 5 hours to about 5 days, about 5 hours to about 4 days, about 5 hours to about 3 days, about 5 hours to about 60 hours, about 5 hours to about 48 hours, about 5 hours to about 36 hours, about 5 hours to about 24 hours,about 5 hours to about 18 hours, about 5 hours to about 12 hours, about 5 hours to about 8 hours, about 5 hours to about 6 hours, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 60 hours, about 6 hours to about 48 hours, about 6 hours to about 36 hours, about 6 hours to about 24 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 8 hours, about 8 hours to about 7 days, about 8 hours to about 6 days, about 8 hours to about 5 days, about 8 hours to about 4 days, about 8 hours to about 3 days, about 8 hours to about 60 hours, About 8 hours to about 48 hours, about 8 hours to about 36 hours, about 8 hours to about 24 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 12 hours to about 18 hours, about 18 hours to about 7 days, about 18 hours to about 6 days, about 18 hours to about 5 days, about 18 hours to about 4 days, about 18 hours to about 3 days, about 18 hours 24 hours to about 60 hours, about 18 hours to about 48 hours, about 18 hours to about 36 hours, about 18 hours to about 24 hours, about 24 hours to about 7 days, about 24 hours to about 6 days, about 24 hours to about 5 days, about 24 hours to about 4 days, about 24 hours to about 3 days, about 24 hours to about 60 hours, about 24 hours to about 48 hours, about 24 hours to about 36 hours, about 36 hours to about 7 days, about 36 hours to about 6 days, about 36 hours to about 5 days, about 36 hours to about 4 days, about 36 hours to about 3 days, about 36 hours to about 60 hours, about 36 hours to about 48 hours, about 48 hours to about 7 days, about 48 hours The antibody is incubated with the antigen for about 48 hours to about 6 days, about 48 hours to about 5 days, about 48 hours to about 4 days, about 48 hours to about 3 days, about 48 hours to about 60 hours, about 60 hours to about 7 days, about 60 hours to about 6 days, about 60 hours to about 5 days, about 60 hours to about 4 days, about 60 hours to about 3 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 7 days, about 5 days to about 6 days, or about 6 days to about 7 days.

[0067] In certain cases, blood samples and / or PBMCs isolated therefrom may be incubated with an antigen (e.g., an antigen specific to an infectious disease or cancer) to promote lymphocyte blastogenesis prior to preparation of microscope slides.

[0068] Antibody labeling The blood sample and / or cells isolated therefrom (e.g., PBMCs) can be contacted with (e.g., labeled with) one or more labeled antibodies (e.g., prior to further processing, such as preparation of a microscope slide). The labeled antibodies can specifically bind to one or more cell surface proteins (e.g., cell surface antigens). For example, the blood sample and / or PBMCs isolated therefrom can be contacted with one or more labeled antibodies, where the labeled antibodies specifically bind to one or more cell surface proteins (e.g., cell surface antigens), such as CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD25, CD27, CD28, CD38, CD39, CD45, CD45RA, CD45RO, CD56, CD57, CD62L, CD66b, CD94, CD103, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD45, CD45RA, CD45RO, CD56, CD57, CD62L, CD66b, CD94, CD103, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139 ... , CD127, CD161, CD223, CD294, CCR4, CCR6, CCR7, CXCR3, CXCR5, HLA-DR, IgA, IgD, IgE, IgG, IgM, TCRγδ, KIR, NKG2A, NKGD, NKp30, NKp44, NKp46, NKp80, CTLA-4, GITR, IgA, IgD, IgE, IgG, and / or IgM. The labeled antibodies may be useful in immunophenotyping blood samples and / or cells isolated therefrom (e.g., PBMCs), for example, by microscopy (e.g., by immunofluorescence microscopy, immunohistochemistry microscopy, immunoelectron microscopy, etc.).

[0069] Antibodies for use in the methods of the invention may be labeled with fluorophores (e.g., fluorescently labeled), enzymes (e.g., enzyme labeled), biotin, magnetic beads, agarose beads, magnetic agarose beads, and / or colloidal gold. For example, antibodies for use in the methods of the invention may be labeled with hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, 3-hydroxyisonicotinaldehyde, Lucifer Yellow, NBD ([2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium; NBD-TMA), R-phycoerythrin (PE), PE-Cy5 conjugates (Cychrome, R670, Tri-Color, Quantum Red), PE-Cy7 conjugates, Red 613 (PE-Texas Red 613), or other conjugates. Red), PerCP (peridinin chlorophyll protein), TruRed (PerCP-Cy5.5 conjugate), FluorX, fluorescein (FITC), BODIPY-FL, G-Dye100, G-Dye200, G-Dye300, G-Dye400, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine, rhodamine B, Texas Red, allophycocyanin (APC), and / or APC-Cy7 conjugate (Far Red).

[0070] In some embodiments, fluorophore-labeled antibodies can be used for detection of cells (e.g., immunophenotyping of cells) by flow cytometry, immunofluorescence staining (e.g., for detection by fluorescence microscopy), or fluorescent Western assays, etc. To detect fluorescent labels, an instrument is required that emits a specified wavelength of light that excites the fluorophore. The fluorescent dye then emits a signal at a different wavelength. The same instrument contains appropriate filters to detect the emission from the fluorophore. Flow cytometry requires the use of a flow cytometer, whereas immunofluorescence (IF) uses a fluorescent microscope. For Western blots, digital imaging systems are typically employed. Antibodies can be labeled with a variety of fluorescent dyes with different excitation and emission spectra. In addition to being highly quantitative, fluorescent labels confer the distinct advantage of being multiplexed, i.e., capable of detecting two or more different target proteins simultaneously, through the use of dyes with non-overlapping emission spectra. For labeling, fluorescent tags (e.g., fluorophores) can be covalently attached to antibodies through primary amine or thiol groups.

[0071] Additionally or alternatively, the antibodies for use in the methods of the present invention may be labeled with enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, or β-galactosidase. Enzyme-labeled antibodies may be used for cell detection (e.g., cell immunophenotyping) by ELISA, Western blotting, immunostaining, and the like. To use enzyme-labeled antibodies, a sample (e.g., a blood sample and / or cells isolated therefrom) may be incubated with an enzyme-specific substrate that is catalyzed by the enzyme to produce a colored product (chromogenic assay) or light (chemiluminescent assay). Each enzyme has a set of substrates and detection methods that may be employed. For example, HRP may react with diaminobenzidine to produce a brown colored product, or with luminol to produce light. In contrast, AP can react with para-nitrophenyl phosphate (pNPP) to produce a yellow-colored product that is detected by a spectrophotometer, or with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) to produce a purple-colored precipitate. Color-producing assays are useful for Western blots, ELISAs, and immunohistochemistry, but light-producing reactions are most frequently used in Western blots. Many substrates with various sensitivities and outputs are available for detection, making enzyme reporters one of the most popular antibody labels.

[0072] Additionally, or alternatively, antibodies for use in the methods of the invention may be labeled with biotin. Biotin is a small molecule (244.3 Da) that forms one of the strongest non-covalent interactions found in nature with its binding partners avidin (found in egg white) and streptavidin (produced by the bacterium Streptomyces avidinii). Biotin rarely interferes with the activity of antibodies due to its size, making it a good choice for labeling. Biotin-labeled antibodies may be used for detection of cells (e.g., immunophenotyping of cells) by Western blot, ELISA, flow cytometry, immunofluorescence, and immunohistochemistry, etc. Biotin-labeled antibodies are often used to increase the sensitivity of assays when antigens are difficult to detect. The blot or sample (e.g., a blood sample and / or cells isolated therefrom) can be incubated with a biotin-labeled antibody, followed by a second incubation with avidin or streptavidin labeled with an enzyme or fluorescent dye. The antibody can be conjugated with multiple biotin molecules (3-6 molecules), which provides an amplification step that enhances the detection of less abundant antigens.

[0073] Preparation of microscope slides The blood sample and / or cells isolated therefrom (e.g., PBMCs) may be placed on a glass slide (e.g., a microscope slide) for evaluation. In certain cases, the blood sample and / or cells isolated therefrom may be contacted with (e.g., labeled with) one or more labeled antibodies prior to placing the cells on the microscope slide. Alternatively, the microscope slide prepared by the method of the present invention may be stained with the labeled antibodies described above. The microscope slide for use in the method of the present invention may be prepared by placing the blood sample and / or cells on the slide by techniques such as direct smear, touch prep, or filter techniques. Additionally or alternatively, the microscope slide for use in the method of the present invention may be prepared by placing the blood sample and / or cells on the slide by using centrifugation, for example, by Cytospin technique. Cytospin preparations may be obtained by employing centrifugal force to isolate, concentrate, and place a monolayer of cells from a dilute cell suspension in a circular area on the slide. The purpose of the Cytospin is to keep the cells intact, thus allowing the cell morphology to be examined. Methods for Cytospin preparations have been described in the literature. See, e.g., Bibby, "Preparation of Cytospin Slides from Bloody Fluids," Lab Med. 17(4):228 (1986). Provided herein are exemplary methods for preparing microscope slides with Cytospin. Briefly, 10 5A number of cells (e.g., cells from a blood sample) can be washed twice with 2% cold FCS-PBS and diluted with 100 μL of 1% cold BSA-PBS. With the cardboard filter facing the center of the cytospin, the slide and filter can be placed in the appropriate slot in the Cytospin device. If there are few cells available, approximately 100 μL of 1% cold BSA-PBS can be dispensed into each well and spun for 1-2 minutes, which serves to wet the filter and allow more cells to reach the slide. Approximately 100 μL of each sample can be dispensed into the appropriate well of the Cytospin and centrifuged at maximum speed for 1-3 minutes. The filters can then be removed from their slides without contacting the smeared specimen on the slide. Slides can be dried overnight in a dehydration chamber before analysis under a microscope and / or further processing. Slides can be fixed (e.g., with cold (4 °C) methanol) immediately after drying. In some cases, the slide can be cover slipped to prepare a permanent slide that can be stored and later evaluated.

[0074] The microscope slide prepared by the above-mentioned method can be examined (e.g., observed and analyzed) under a microscope to determine the number of viable mononuclear blast cells and / or the number of viable large granular lymphocytes in the slide. The number or level of viable mononuclear blast cells and / or the number of viable large granular lymphocytes in a blood sample can be determined based on the number of viable mononuclear blast cells and / or the number of viable large granular lymphocytes in the microscope slide.

[0075] Prognostic assessment for cancer and / or infectious diseases Subjects whose blood sample (and / or cells prepared from the blood sample) exhibits an increased number or level of viable mononuclear blast cells and / or an increased number or level of viable large granular lymphocytes compared to a reference level (e.g., any of the exemplary reference levels described herein) may be identified as having an improved prognosis of cancer and / or infectious disease. For example, an increased number or level (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, an increase of at least 280%, at least 300%, at least 320%, at least 340%, at least 360%, at least 380%, or at least 400%, or between about a 1% increase and about a 400% increase (or any sub-ranges of this range described herein)) of viable mononuclear blasts, and / or an increased number or level (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase,Subjects having at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, at least a 320% increase, at least a 340% increase, at least a 360% increase, at least a 380% increase, or at least a 400% increase, or between about a 1% increase and about a 400% increase (or any sub-range of this range described herein)) of viable large granular lymphocytes may be identified as having an improved prognosis of cancer and / or infectious disease.

[0076] Subjects having a decreased number or level of viable mononuclear blasts and / or a decreased number or level of viable large granular lymphocytes compared to a reference level (e.g., any of the exemplary reference levels described herein) may be identified as having a poor prognosis for one or more cancers and / or infectious diseases. For example, a decreased number or level (e.g., at least a 1% decrease, at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, at least a 95% decrease, or at least a 99% decrease, or between about a 1% decrease and about a 99% decrease (or any sub-ranges of this range described herein)) of viable mononuclear cell blasts as compared to a reference level (e.g., any of the exemplary reference levels described herein). Subjects having a reduced number or level (e.g., at least a 1% reduction, at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, or from about a 1% reduction to about a 99% reduction (or any sub-range of this range described herein)) of viable large granular lymphocytes may be identified as having a poor prognosis for cancer and / or infectious disease.

[0077] In certain instances, the reference level is about the 70th percentile, about the 71st percentile, about the 72nd percentile, about the 73rd percentile, about the 74th percentile, about the 75th percentile, about the 76th percentile, about the 77th percentile, about the 78th percentile, about the 79th percentile, about the 80th percentile, about the 81st percentile, about the 82nd percentile, about the 83rd percentile, about the 84th percentile, about the 85th percentile, about the 86th percentile, about the 87th percentile, about the 88th percentile, about the 89th percentile, about the 90th percentile, about the 91st percentile, about the 92nd percentile, about the 93rd percentile, about the 94th percentile, about the 95th percentile, about the 96th percentile, about the 97th percentile, about the 98th percentile, about the 99th percentile, about the 80th percentile, about the 98th percentile, about the 99th percentile, about the 100th percentile, about the 101st percentile, about the 102nd percentile, about the 103rd percentile, about the 104th percentile, about the 105th percentile, about the 106th percentile, about the 107th percentile, about the 108th percentile, about the 109th percentile, about the 110th percentile, about the 111th percentile, about the 112th percentile, about the 113th percentile, about the 114th percentile, about the 115th percentile, about the 116th percentile, about the 117th percentile, about the 118th percentile, about the 119th percentile, about the 120 The percentile may be about the 82nd percentile, about the 83rd percentile, about the 84th percentile, about the 85th percentile, about the 86th percentile, about the 87th percentile, about the 88th percentile, about the 89th percentile, about the 90th percentile, about the 91st percentile, about the 92nd percentile, about the 93rd percentile, about the 94th percentile, about the 95th percentile, about the 96th percentile, about the 97th percentile, about the 98th percentile, or about the 99th percentile). A healthy patient population can be a patient population that is not currently diagnosed with cancer and / or an infectious disease or has not been diagnosed with cancer and / or an infectious disease within the past 5 years (e.g., within the past 4 years, within 3 years, within 2 years, within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, or within 1 week).

[0078] The subject identified by the method of the present invention as having improved or poor prognosis of cancer and / or infectious disease can be treated accordingly.For example, the subject identified as having improved prognosis of cancer and / or infectious disease can be selected for low-frequency screening of cancer and / or infectious disease.Low-frequency screening can include screening once every year, every two years, every three years, every four years, every five years, every six years, every seven years, every eight years, every nine years, every ten years, or more than once every ten years.The subject identified as having poor prognosis of cancer and / or infectious disease can be selected for high-frequency screening of cancer and / or infectious disease. Frequent screening may include screening once per day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, or every 11 months. Frequent screening may include screening at least twice a year, screening at least three times a year, screening at least four times a year, screening at least five times a year, screening at least six times a year, screening at least seven times a year, screening at least eight times a year, screening at least nine times a year, screening at least ten times a year, screening at least 11 times a year, screening at least 12 times a year, screening at least 24 times a year, screening at least 52 times a year (e.g., weekly screening), or screening at least 365 times a year (e.g., daily screening).

[0079] subject As used herein, the term "subject" can refer to humans and non-human animals. Non-human animals can include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cows, chickens, dogs, mice, rats, goats, rabbits, and pigs. For example, the subject can be a human.

[0080] In certain cases, the subject has not been identified or diagnosed with cancer and / or an infectious disease. For example, the subject may not currently (or recently) be identified or diagnosed with cancer and / or an infectious disease, for example, within the past 5 years (e.g., within the past 4 years, within the past 3 years, within the past 2 years, within the past year, within the past 11 months, within the past 10 months, within the past 9 months, within the past 8 months, within the past 7 months, within the past 6 months, within the past 5 months, within the past 4 months, within the past 3 months, within the past 2 months, within the past month, within the past 3 weeks, within the past 2 weeks, or within the past week).

[0081] Other Aspects While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for determining the prognosis of cancer or an infectious disease in a subject, comprising the steps of: (a) contacting a blood sample obtained from said subject with a cell viability dye; (b) after step (a), preparing a microscope slide using the blood sample; (c) using microscopic analysis to determine the number of viable mononuclear cell blasts and / or the number of viable large granular lymphocytes in the microscope slide; and (d) identifying subjects having an increased number of viable mononuclear cell blasts and / or an increased number of viable large granular lymphocytes compared to the reference level as having an improved prognosis for cancer or infectious disease; or Identifying subjects having a decreased number of viable mononuclear cell blasts and / or a decreased number of viable large granular lymphocytes compared to the reference levels as having a poor prognosis for cancer or infectious disease.

2. The method of claim 1, wherein the subject has never been identified or diagnosed as having cancer or an infectious disease.

3. A method for selecting a treatment for a subject who has not been identified or diagnosed as having cancer or an infectious disease, comprising the steps of: (a) contacting a blood sample obtained from said subject with a cell viability dye; (b) after step (a), preparing a microscope slide using the blood sample; (c) using microscopic analysis to determine the number of viable mononuclear cell blasts and / or the number of viable large granular lymphocytes in said microscope slide; (d) identifying subjects having an increased number of viable mononuclear cell blasts and / or an increased number of viable large granular lymphocytes compared to the reference level as having an improved prognosis for cancer or infectious disease; or Identifying subjects having a decreased number of viable mononuclear cell blasts and / or a decreased number of viable large granular lymphocytes compared to a reference level as having a poor prognosis for cancer or infectious disease; and (e) selecting a subject identified as having an improved prognosis for cancer or infectious disease for infrequent screening for cancer or infectious disease; or For subjects identified as having a poor prognosis for cancer or infectious disease, selecting the subject for frequent screening for cancer or infectious disease.

4. 4. The method of any one of claims 1 to 3, wherein step (b) comprises one or more centrifugation steps.

5. 5. The method of claim 4, wherein one of the centrifugation steps comprises the use of a Cytospin device.

6. 4. The method of any one of claims 1 to 3, wherein step (c) comprises determining the number of viable mononuclear cell blasts in the microscope slide.

7. 4. The method of any one of claims 1 to 3, wherein step (c) comprises determining the number of viable large granular lymphocytes in the microscope slide.

8. 4. The method of any one of claims 1 to 3, wherein step (c) comprises determining the number of viable mononuclear blast cells and the number of viable large granular lymphocytes in the microscope slide.

9. 4. The method of any one of claims 1 to 3, wherein step (b) comprises the use of one or more fluorophore-labeled antibodies specific for one or more cell surface proteins.

10. 10. The method of claim 9, wherein the one or more labeled antibodies specifically bind to an antigen selected from the group consisting of CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD25, CD27, CD28, CD38, CD39, CD45, CD45RA, CD45RO, CD56, CD57, CD62L, CD66b, CD94, CD103, CD122, CD123, CD127, CD161, CD223, CD294, CCR4, CCR6, CCR7, CXCR3, CXCR5, IgA, IgD, IgE, IgG, IgM, TCRγδ, KIR, NKG2A, NKGD, NKp30, NKp44, NKp46, NKp80, CTLA-4, and GITR.

11. 4. The method of claim 1, further comprising, prior to step (b), incubating the blood sample with an antigen specific to the infection for 6 hours to 3 days.

12. The method of any one of claims 1 to 3, wherein the viable mononuclear cell blasts are viable T cell blasts and / or viable B cell blasts.

13. The method of any one of claims 1 to 3, wherein the viable large granular lymphocytes are NK cells.

14. The method of any one of claims 1 to 3, wherein the blood sample is a venous blood sample.

15. 4. The method of any one of claims 1 to 3, wherein the blood sample is a capillary blood sample.

16. 16. The method of claim 15, wherein the capillary blood sample is a fingerstick blood sample.

17. 4. The method of any one of claims 1 to 3, further comprising the step of producing a frozen blood sample from a portion of the blood sample.

18. thawing the frozen blood sample; and performing additional immunological tests / analyses on the thawed blood samples; 18. The method of claim 17, further comprising:

19. The method of any one of claims 1 to 3, comprising selecting, for a subject identified as having an improved prognosis for cancer or infectious disease, for less frequent screening for cancer or infectious disease.

20. The method of any one of claims 1 to 3, comprising a step of selecting a subject identified as having a poor prognosis for cancer or infectious disease for frequent screening for cancer or infectious disease.

Images