Non-invasive cell-based blood biopsy

Abstract

A noninvasive blood-based biopsy measures surface protein receptors on specific circulating cell populations. Dysregulated quantities of specific surface protein receptors on specific circulating cells indicate the diseased tissue origin. Thus, specific circulating cells serve as proxies for diseased tissue cells as they are shed from such tissues. This approach can be used not only for preeclampsia but also for other vascular disorders where detecting surface protein receptors associated with particular disorders can increase sensitivity and disease specificity. Detection of a high level VEGFR1 on isolated circulating cells that are immunopositive for CD34 and CD31 can be used to identify and treat subjects suffering from preeclampsia.

Description

NON-INVASIVE CELL-BASED BLOOD BIOPSY

[0001] This application claims benefit of United States provisional patent application number 63 / 639,373, filed April 26, 2024, the entire contents of which are incorporated by reference into this application. ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No.7R01HL159946- 02, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Preeclampsia is responsible for over 70,000 maternal and 500,000 fetal deaths annually worldwide. Early screening and intervention are critical for preventing and monitoring preeclampsia. However, current screening methods, such as monitoring blood pressure, proteinuria, maternal symptoms, and serum biomarkers, lack placental specificity and are affected by systemic vascular stress, offering modest sensitivity in identifying and predicting preeclampsia. The existing blood tests applied to rule out preeclampsia (e.g., a high placental growth factor (PlGF) measurement at 1st and 2nd trimesters and a soluble VEGFR1 / PlGF ratio of ≤ 38 during 3rd trimester) have limited sensitivity in predicting preeclampsia. Moreover, this association does not hold for all patients. Some patients who develop late-stage preeclampsia have very high PlGF at early times. Thus, "low" PlGF is one early marker that is associated with preeclampsia, but does not provide a reliable proxy. Therefore, enhancing the accuracy and sensitivity of early preeclampsia diagnosis is vital to protecting maternal and fetal health. This problem with preeclampsia is representative of vascular disorders in which greater accuracy can be achieved by detecting surface protein receptors, such as vascular endothelial growth factor receptor (VEGFR), associated with particular disorders when their levels are dysregulated on diseased tissue cells.

[0004] There remains a need for a non-invasive biopsy approach to identify the dysregulated surface protein receptors exhibited by the diseased tissue cells. These cell receptors offer more precise indications of the disorder compared to systemic biomarkers like blood pressure and serum protein levels. SUMMARY

[0005] The need is met by measuring surface protein receptors on specific circulating cell populations. Dysregulated quantities of specific surface protein receptors on specific circulating cells indicate the diseased tissue origin. Thus, specific circulating cells serve asproxies for diseased tissue cells as they are shed from such tissues. This approach can be used not only for preeclampsia but also for other vascular disorders where detecting surface protein receptors associated with particular disorders can increase sensitivity and disease specificity.

[0006] The material described herein meets these needs and others by providing a method of detecting a disorder in a subject, the method comprising: (a) isolating circulating tissue- origin cells from a blood sample obtained from the subject; and (b) measuring the amount of surface protein receptors per circulating tissue-origin cell. The disease status is identified by comparing the measured amount and distribution of receptors to the established healthy reference and pathological thresholds of particular disorders. A disorder is detected when the amount of surface protein receptors per circulating tissue-origin cell is greater than a reference amount.

[0007] In some embodiments, the disorder is a vascular disorder. In some embodiments, the vascular disorder is preeclampsia. In some embodiments, the circulating cells to be isolated comprise cells that are immunopositive for CD34 and CD31. In some embodiments, these cells are circulating progenitor cells (cPCs). In some embodiments, the circulating progenitor cells are circulating endothelial cells (cECs). In some embodiments, the surface protein receptors are VEGFR1. In some embodiments for detection of preeclampsia, the circulating tissue-origin cells are immunopositive for CD34 and CD31, and preeclampsia is detected when a geometric mean of more than 200 VEGFR1 per cell are measured. In some embodiments, preeclampsia is detected when at least 236 VEGFR1 per cell are measured. Values above 236 VEGFR1 / cell indicate severe PE. In some embodiments, the reference value is the geometric mean of the entire VEGFR measurement distribution. In some embodiments, the reference value is the median value of the entire VEGFR measurement distribution.

[0008] In some embodiments, detection is based on quantifying the percentage of cells presenting a specific amount of VEGFR that differentiate disease states from healthy controls. In some embodiments for detection of preeclampsia, the surface protein receptors are vascular endothelial growth factor receptors (VEGFRs), the circulating tissue-origin cells are circulating endothelial cells (cECs), and preeclampsia is detected when high-VEGFR cECs are present. In some embodiments, high-VEGFR cECs is greater than 60% of cECs. In some embodiments, preeclamptic cECs have at least 150,000 VEGFR1 and at least 10,000 VEGFR2 per cell. In embodiments such as this, the reference amount for a high- VEGFR cEC population is 45% with 138,000 VEGFR1 / cell, and half of this high-VEGFR population has a moderate-VEGFR2 amount of 39,100 VEGFR2 / cell and the other half has a high-VEGFR2 amount of 236,000 VEGFR2 / cell (See, e.g., FIG.4D). The cECs are thoseshed from preeclamptic vessels. In some embodiments, the surface protein receptor is VEGFR1. In some embodiments, the population of cECs exhibits 200,000 or more VEGFR1 per cEC.Preeclampsia refers to the new onset of hypertension and proteinuria or the new onset of hypertension plus significant end-organ dysfunction with or without proteinuria in a previously normotensive patient, typically after 20 weeks of gestation or postpartum. Several subtypes of preeclampsia exist, with a variety of pathophysiological pathways leading to maternal and fetal mortality and morbidity. The most commonly described subtypes are early onset (<34 weeks of gestation) and late onset (≥34 weeks of gestation). The clinical features overlap, but the spectrum of disease and outcomes differ: Early-onset disease has been associated with more severe placental and maternal / fetal clinical findings and, in turn, poorer maternal / fetal outcomes.

[0009] In some embodiments, the subject, with respect to the methods described herein, is 8-34 weeks pregnant. In some embodiments, the subject is 8-12 weeks pregnant. In some embodiments, the subject is approximately 16 weeks pregnant. In some embodiments, the subject is 8-20 weeks pregnant. In some embodiments, the subject is about 26 weeks pregnant. In some embodiments, the subject is 8-26 weeks pregnant. In some embodiments, the subject is post-partum. As is understood by those skilled in the art, the length of pregnancy, or gestational age, is based on the date of the last menstrual period.

[0010] Also described herein is a method of measuring the amount of VEGFR present in preeclamptic, placental or endometrial vasculature or other tissue vasculature that is affected by preeclampsia in a subject. In some embodiments, the method comprises: (a) isolating circulating endothelial cells (cECs) from a blood sample obtained from the subject; and (b) measuring the amount of vascular endothelial growth factor receptors (VEGFRs) on the cECs per cEC. The amount of VEGFRs per cell can be compared to a reference value, wherein differences from the reference value is indicative of a vascular disorder. In some embodiments, the VEGFRs comprise VEGFR1. In some embodiments, the VEGFRs further comprise VEGFR2. In some embodiments, the surface receptor proteins comprise other plasma membrane proteins or combination of VEGFR and other plasma membrane proteins.

[0011] Preeclampsia status is identified by comparing the measurements to the established uncomplicated pregnant reference (low-VEGFR cEC population (55%): 3000 VEGFR1 / cell and 3000 VEGFR2 / cell; high-VEGFR cEC population (45%): 138,000 VEGFR1 / cell and 39,100-236,000 VEGFR2 / cell) and, optionally, preeclamptic thresholds (low-VEGFR population (< 40%): 3,000 VEGFR1 / cell and 3000 VEGFR2 / cell; high-VEGFR population (> 60%): 150,000 VEGFR1 / cell and 10,000 VEGFR2 / cell). The amount of VEGFR relative to the amount of cECs is indicative of the amount of VEGFR present in preeclamptic vasculature of the subject.

[0012] In some embodiments, the cECs are immunopositive for CD34. In some embodiments, the cECs are immunopositive for CD34 and CD31. In some embodiments, the cECs are immunopositive for CD34, CD31, and CD146. In some embodiments, the cECs are immunopositive for von Willebrand factor (vWF), CD105, and CD144. In some embodiments, the cECs are immunonegative for CD45. In some embodiments, the isolating comprises cell capture via contacting the sample with an antibody to human CD34. In some embodiments, the isolating comprises cell capture via contacting the sample with any of the aforementioned immunopositive antibodies. In some embodiments, the isolating comprises cell capture via excluding from the sample cECs identified with any of the aforementioned immunonegative antibodies.

[0013] The methods described herein typically comprise quantitative flow cytometry as the method of measuring. Suitable methods of measuring provide a means to measure the amount of surface protein receptors per circulating cell. By isolating cells of a specific type, e.g. based on tissue origin and disease type, it is possible to determine the amount of receptor per cell of that type, providing for a more specific, accurate, and meaningful assay.

[0014] Also described is a method of treating a subject for preeclampsia. In some embodiments, the method comprises: (a) performing the method described above on a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, and / or blood pressure monitoring when the cECs consist of a low-VEGFR population (< 40%, 3,000 VEGFR1 / cell and 3000 VEGFR2 / cell) and a high-VEGFR population (> 60%, 150,000 VEGFR1 / cell and 10,000 VEGFR2 / cell). In some embodiments, the method of treating a human subject for preeclampsia comprises: (a) measuring at least 200,000 VEGFR1 per cEC in cECs isolated from a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, and / or blood pressure monitoring. In some embodiments, the method further comprises measuring at least 10,000 VEGFR2 per cEC.

[0015] In some embodiments, the method of treating a human subject for preeclampsia comprises: (a) measuring a geometric mean of at least 200, or about 236 VEGFR1 per circulating CD34+CD31+ cells isolated from a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, blood pressure monitoring, and / or plan for early induction of labor. In some embodiments, the isolated cells are circulating progenitor cells (cPCs). In some embodiments, the cPCs are circulating endothelial cells (cECs).

[0016] Detection of preeclampsia early in pregnancy, e.g., during the first trimester, or before 20 weeks, allows for treatment with prophylactic aspirin therapy (low-dose aspirin).Other treatments for preeclampsia, typically for more severe cases, include administration of anti-hypertensive medications to lower blood pressure, anticonvulsant medication, such as magnesium sulfate, to prevent seizures, and corticosteroids to promote development of the baby's lungs before delivery. In pregnancy, high blood pressure is diagnosed if the systolic pressure is 140 millimeters of mercury (mm Hg) or higher or if the diastolic pressure is 90 mm Hg or higher.

[0017] In some embodiments, the reference amount of surface protein receptors is measured in a healthy control subject. In some embodiments, the subject is a human. In some embodiments, the blood sample comprises 10-20 ml blood. In some embodiments, the blood sample comprises 6-15 ml blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS.1A-1F. Circulating progenitor cells (cPCs) and circulating endothelial cells (cECs) were identified in the pre enriched CD34+ cell population. (1A) Debris and small platelets (≤ 6μm) were excluded from the pre-enriched CD34+ cells. (1B) Cell singlets were selected based on cell width (FSC-Width). (1C) cPCs were identified as CD34+ CD31+ cells. (1D) cECs were identified as CD34+ CD31+ CD146+ and were rare in the healthy blood samples, comprising 0.98 ± 0.29 % of the cPC population (n=23, mean ± SEM). (1E, 1F) Fluorescence controls (i.e., FMO) of APC-Cy7-CD31 and APC-CD146 were used to set the respective positive gates.

[0019] FIGS.2A-2F. Quantitative cell-by-cell variations of VEGFR expression by CD34+CD31+ cPCs and CD34+CD31+CD146+ cECs in 23 healthy blood samples. The histograms are ranked by median VEGFR concentrations from low to high (color maps). (2A) Cell-by-cell distributions of VEGFR1 and (2B) VEGFR2 on cPCs in 23 individuals’ blood (age 46 ± 11, 11 females and 12 age-matched males). (2C) Median levels of VEGFR1 and VEGFR2 on cPCs were extracted from the frequency histograms. (2D) Frequency histograms of VEGFR1 and (2E) VEGFR2 on cECs in 23 individuals’ blood. (2F) Median levels of VEGFR1 and VEGFR2 on cECs were extracted from the frequency histograms. Dashed lines indicate the previously reported ensemble averages of VEGFR1 and VEGFR2 concentrations of human umbilical vascular endothelial cells, in vitro (800 VEGFR1s and 1800 VEGFR2s per HUVEC). Subject codes are formatted as age (sex).

[0020] FIGS.3A-3B. VEGFR1 plasma membrane localization in cPCs is detected in menopausal / postmenopausal females, not in peri / premenopausal females. (3A) The numbers of VEGFR1s / cPC of individual female samples are calculated as the populationmedians of each cell sample (10,000 cPCs per sample). The dashed line indicates the previously established detection threshold (sample median, 500 VEGFRs / cell). The VEGFR1 measurements are under the detection threshold for all the peri / premenopausal females. The error bars represent mean ± SE (P = 0.2). (3B) Pooled VEGFR1 measurements represent the difference in the VEGFR1 distribution on cPCs between females over 50 y / o (n=5) and females under 50 y / o (n=6). The population medians of VEGFR1 concentrations are 88 VEGFR1s / cPC and 650 VEGFR1s / cPC in pooled peri / premenopausal females and menopausal / postmenopausal females, respectively.

[0021] FIGS 4A-4F. Peri / premenopausal females have prominent VEGFR-intermediate / high cEC subpopulations that menopausal / postmenopausal females (50-60 y / o) lack. Inspired by the age-specific differences in the median VEGFR concentrations on cECs, we separately pooled the VEGFR measurements from (4A-4B) Menopausal / postmenopausal females and (4C-4D) Peri / premenopausal females to compare the VEGFR distributions between these two groups. Each component (Comp) represents a cEC subpopulation identified via mixture modeling analysis. (4A-4B) In menopausal / postmenopausal females, cECs expressed 2,900 VEGFR1 and 3,400 VEGFR2 per cell (median of cell populations). (4C) In peri / premenopausal females, 44.7% cECs made up VEGFR-intermediate / high subpopulations that expressed 138,000 VEGFR1 / cell. (4D) The peri / premenopausal females showed three cEC subpopulations based on median VEGFR2 concentrations: 55.8% cECs had 2,600 VEGFR1 / cell, 21.8% had 39,100 VEGFR2 / cell, and 22.4% had 236,000 VEGFR2 / cell. (4E) Since no age-related difference was statistically identified among males, we pooled cEC measurements from males of all ages and observed two cEC subpopulations based on VEGFR1 densities: 73.1% cECs had 3,100 VEGFR1 / cell and 26.9% had 206,000 VEGFR1 / cell. (4F) Two cEC subpopulations were observed based on VEGFR2 concentrations: 72.4% cECs had 3,000 VEGFR2 / cell and 27.6% had 155,000 VEGFR2 / cell. These cEC subpopulations in healthy individuals were stratified and quantitatively characterized by their median VEGFR concentrations via adapting an established mixture modeling.

[0022] FIG.5. Hypothetical cPC and cEC population compositions in healthy females and males.

[0023] FIG.6. The standardized workflow for establishing VEGFR quantities in circulating angiogenic cells: (1) Enrich CD34+ cells from healthy individuals' whole blood with immunomagnetic beads (IB). (2) Label CD34+ cells with indicated fluorescent antibodies and (3) perform single-cell fluorescence screening with a flow cytometer. (4a) Stratify cEC / cPC via cell marker phenotyping. (4b) Translate PE-VEGFR signal intensity to VEGFR quantity with PE calibration curve. (5a) Quantify VEGFR on cECs and cPCs from healthy individuals'blood samples on a cell-by-cell manner. (5b) Stratify healthy subjects based on correlations between VEGFR quantification and subject characteristics (e.g., sex, age, menopause, etc.). (5c) Pool single-cell measurements from an identified subject group to establish VEGFR distribution profiles for that subject group. We mark common cEC subpopulations presenting in healthy groups in order to help distinguish pathological cEC subpopulations shed from diseased tissues.

[0024] FIG.7. Cell-to-cell VEGFR distributions in cECs of a pregnant woman at her antenatal visit. The described blood test was conducted on a pregnant woman (10 mL whole blood, unknown diagnosis) to demonstrate the technical and statistical capabilities of the test. The test distinguished between a low-VEGFR1 cEC population expressing 2647 VEGFR1 / cell and a high-VEGFR1 cEC population expressing 8000 VEGFR1 / cell. The low- VEGFR2 population expressing 960 VEGFR2 / cell and the high-VEGFR2 population expressing 17,000 VEGFR2 / cell. The grey-shaped plot represents raw data before applying mixture modeling analysis.

[0025] FIGS.8A-8B. Illustrate the confirmation of the test protocol with standards. (8A) Schematic overview of the blood test protocol. Currently the test takes about 4 hours, of which about 1.5 hours is hands-on. (8B) The protocol does not alter biomarker level on the cell standard. To verify the test's accuracy in measuring biomarker levels on these cells, blood samples were spiked with 800 to 80,000 cells / mL of cultured cells. Cells were retrieved using the protocol illustrated in FIG.8A, and their biomarker levels were compared to cells directly from the culture flask. The flow cytometry results show that the protocol retrieves the targeted cells, even at low abundance (800 cells / mL), and measures biomarker levels without altering biomarker expression. Testing was performed using 800 – 80,000 cells / mL.

[0026] FIGS.9A-9B. Demonstration that the test results are reproducible. FIG.9A shows the results of a test performed on a sample obtained at Patient 012’s first visit, at 36 weeks, 5 days, which showed 140 VEGFR1 / cell and 1400 VEGFR2 / cell. FIG.9B shows the results of a test performed on the same patient’s second visit, at 39 weeks, 3 days. This second visit’s test result showed 200 VEGFR1 / cell and 1100 VEGFR2 / cell. This patient did not develop preeclampsia.

[0027] FIGS.10A-10B. P:C ratio test exhibited 63% sensitivity at detecting control versus severe PE (10A), while the VEGFR1 test exhibited 91% sensitivity (10B). The P:C ratio measurements and VEGFR1 measurements were from the same patients.

[0028] FIGS.11A-11B. Scatterplots of VEGFR1 concentration for each patient as a single data point. Twins (2) were excluded from the data. In FIG.11A, superimposed PE, patientswho had hypertension before week 20 (SIPE) are shown separately. In FIG.11B, PE and SIPE are combined.

[0029] FIG.12. Diagnostic performance of the VEGFR1-based test achieved an area under the curve (AUC) of 0.78 (95% CI), with 78% sensitivity at 75% specificity for identifying severe PE. The cutoff value is 236 VEGFR1 per cell, where values above 236 VEGFR1 / cell indicate severe PE, yielding an AUC of 0.76.

[0030] FIGS.13A-13B. Consideration of BMI and VEGFR in PE. Identification of 83% severe PE cases in patients without obesity (5 out of 6) shown in FIG.13A. FIG.13B shows the sensitivity and specificity achieved with VEGFR1 alone and with VEGFR1+BMI. The VEGFR1+BMI test achieved an area under the curve (AUC) of 0.85 (95% CI), with 89% sensitivity at 75% specificity for identifying severe PE.

[0031] FIG.14. Scatterplot of the number of VEGFR2 per cell for control, mild PE, severe PE, and postpartum PE. Twins (2) were excluded. PE and SIPE were combined. DETAILED DESCRIPTION

[0032] The disclosure described herein is based on the discovery of a non-invasive cell- based blood biopsy that assesses membrane VEGFRs on circulating tissue-origin cells, which informs the membrane VEGFR abundance on placental vasculature and has the potential to complement or even replace the current non-specific diagnostic method, leading to improved outcomes for both the mother and the fetus. Moreover, this provides a means for assessing other surface protein receptors on circulating cells that originate from disease- affected tissues. Rather than relying on soluble markers, quantification of the protein levels, or use of vesicles, this method provides an indication of the amount per cell of surface protein receptor present on the cell type of interest from the diseased tissue. Definitions

[0033] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0034] As used herein, a “control” or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects, or a baseline amount of marker to be used for comparison. Typically, a baseline will be a measurement taken from the same or different subject or patient, for example, obtained prior to or earlier in pregnancy of the same patient, or representing an uncomplicated pregnancy. The sample can be an actual sample used fortesting, or a reference level or range, based on known normal measurements of the corresponding marker.

[0035] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the recited embodiment. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.” “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the disclosure herein.

[0036] The term "effective amount" or "therapeutically effective amount" or "prophylactically effective amount", refer to an amount of an active agent described herein that is effective to provide the desired / intended result and / or biological activity. Thus, for example, in various embodiments, an effective amount of a composition described herein is an amount that is effective to result in remission or slowing the progression of disease, and / or to improve or to ameliorate symptoms of and / or to treat disease.

[0037] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results.

[0038] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.

[0039] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.

[0040] As used herein, to “prevent” or “protect against” a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease.

[0041] The term “about,” as used herein when referring to a measurable value such as an amount, level or concentration, for example and without limitation, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount, or fold differences in levels of a quantifiable comparison with a standard or control or referencematerial, such as 1-fold, 2-fold, 3-fold, 4-fold…10-fold, 100-fold, etc. of the specified level of comparison.

[0042] The terms “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose. Methods

[0043] The methods provided herein measure surface protein receptors on specific circulating cell populations. Dysregulated quantities of specific surface protein receptors on specific circulating cells indicate the diseased tissue origin. Thus, specific circulating cells serve as proxies for diseased tissue cells as they are shed from such tissues. This approach can be used not only for preeclampsia but also for other vascular disorders where detecting surface protein receptors associated with particular disorders, such as cancers, can increase sensitivity and disease specificity.

[0044] The material described herein meets these needs and others by providing a method of detecting a disorder in a subject. In some embodiments, the method comprises: (a) isolating circulating tissue-origin cells from a blood sample obtained from the subject; and (b) measuring the amount of surface protein receptors per circulating tissue-origin cell. The disease status is identified by comparing the measured amount and distribution of receptors to the established healthy reference and pathological thresholds of particular disorders. A disorder is detected when the amount of surface protein receptors per circulating tissue- origin cell is greater than a reference amount.

[0045] In some embodiments, the disorder is a vascular disorder. In some embodiments, the vascular disorder is preeclampsia. In some embodiments, the circulating cells to be isolated comprise cells that are immunopositive for CD34 and CD31. In some embodiments, the surface protein receptors are VEGFR1. In some embodiments, preeclampsia is detected when greater than 200 VEGFR1 per isolated cell are measured. In some embodiments, preeclampsia is detected when 236 VEGFR1 or more per isolated cell are measured.

[0046] In some embodiments for detection of preeclampsia, the surface protein receptors are vascular endothelial growth factor receptors (VEGFRs), the circulating tissue-origin cells are circulating endothelial cells (cECs), and preeclampsia is detected when high-VEGFR cECs are present. In some embodiments, high-VEGFR cECs is greater than 60% of cECs. In some embodiments, preeclamptic cECs have at least 150,000 VEGFR1 and at least 10,000 VEGFR2 per cell. In embodiments such as this, the reference amount for a high- VEGFR cEC population is 45%, with 138,000 VEGFR1 / cell and 39,100-236,000VEGFR2 / cell. The cECs are those shed from preeclamptic vessels. In some embodiments, the surface protein receptor is VEGFR1. In some embodiments, the population of cECs exhibits 200,000 or more VEGFR1 per cEC.

[0047] Preeclampsia refers to the new onset of hypertension and proteinuria or the new onset of hypertension plus significant end-organ dysfunction with or without proteinuria in a previously normotensive patient, typically after 20 weeks of gestation or postpartum. Several subtypes of preeclampsia exist, with a variety of pathophysiological pathways leading to maternal and fetal mortality and morbidity. The most commonly described subtypes are early onset (<34 weeks of gestation) and late onset (≥34 weeks of gestation). The clinical features overlap, but the spectrum of disease and outcomes differ: Early-onset disease has been associated with more severe placental and maternal / fetal clinical findings and, in turn, poorer maternal / fetal outcomes.

[0048] In some embodiments, the subject, with respect to the methods described herein, is 8-34 weeks pregnant. In some embodiments, the subject is 8-12 weeks pregnant. In some embodiments, the subject is approximately 16 weeks pregnant. In some embodiments, the subject is 8-20 weeks pregnant. In some embodiments, the subject is about 26 weeks pregnant. In some embodiments, the subject is 8-26 weeks pregnant. In some embodiments, the subject is post-partum. As is understood by those skilled in the art, the length of pregnancy, or gestational age, is based on the date of the last menstrual period.

[0049] Also described herein is a method of measuring the amount of VEGFR present in placental or endometrial vasculature or other tissue vasculature that is affected by preeclampsia in a subject. Such tissue is referred to herein as preeclamptic vasculature. In some embodiments, the method comprises: (a) isolating circulating endothelial cells (cECs) from a blood sample obtained from the subject; and (b) measuring the amount of vascular endothelial growth factor receptors (VEGFRs) on the cECs per cEC. The amount of VEGFRs per cell can be compared to a reference value, wherein differences from the reference value is indicative of a vascular disorder. In some embodiments, the VEGFRs comprise VEGFR1s. In some embodiments, the VEGFRs further comprise VEGFR2s.

[0050] Preeclampsia status is identified by comparing the measurements to the established uncomplicated pregnant reference (low-VEGFR cEC population (55%): 3000 VEGFR1 / cell and 3000 VEGFR2 / cell; high-VEGFR cEC population (45%): 138,000 VEGFR1 / cell and 39,100-236,000 VEGFR2 / cell) and, optionally, preeclamptic thresholds (low-VEGFR population (< 40%): 3,000 VEGFR1 / cell and 3000 VEGFR2 / cell; high-VEGFR population (> 60%): 150,000 VEGFR1 / cell and 10,000 VEGFR2 / cell). The amount of VEGFR relative tothe amount of cECs is indicative of the amount of VEGFR present in preeclamptic vasculature of the subject.

[0051] While terms such as “progenitor” and “endothelial” have been used to describe cells with a range of marker profiles and functional tests, for the purposes herein, it is recognized that circulating endothelial cells (cECs) represent a subset of circulating progenitor cells (cPCs). The markers CD34 and CD31 are associated with cPCs and also present on cECs. The circulating cells to be isolated for use in the methods described herein are immunopositive for CD34 and CD31. In some embodiments, the isolated cells are immunopositive for additional markers, such as CD146.

[0052] In some embodiments, the cECs are immunopositive for CD34. In some embodiments, the cECs are immunopositive for CD34 and CD31. In some embodiments, the cECs are immunopositive for CD34, CD31, and CD146. In some embodiments, the cECs are immunopositive for vWF, CD105, and CD144. In some embodiments, the cECs are immunonegative for CD45. In some embodiments, the isolating comprises cell capture via contacting the sample with an antibody to human CD34. In some embodiments, the isolating comprises cell capture via contacting the sample with any of the aforementioned immunopositive antibodies. In some embodiments, the isolating comprises cell capture via excluding from the sample cECs identified with any of the aforementioned immunonegative antibodies.

[0053] The methods described herein typically comprise quantitative flow cytometry as the method of measuring. Suitable methods of measuring provide a means to measure the amount of surface protein receptors per circulating cell. By isolating cells of a specific type, e.g. based on tissue origin and disease type, it is possible to determine the amount of receptor per cell of that type, providing for a more specific, accurate, and meaningful assay.

[0054] In some embodiments, organic fluorophore-conjugated antibodies, such as Phycoerythrin-conjugated VEGFR antibodies, are used to label the cells for flow cytometry analysis. In some embodiments, the fluorophore molecules are quantum dots or customized small molecules, such as aptamers.

[0055] Also described is a method of treating a subject for preeclampsia. In some embodiments, the method comprises: (a) performing the method described above on a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, and / or blood pressure monitoring when the cECs consist of a low-VEGFR population (< 40%, 3,000 VEGFR1 / cell and 3000 VEGFR2 / cell) and a high-VEGFR population (> 60%, 150,000 VEGFR1 / cell and 10,000 VEGFR2 / cell). This number is based on the finding that peri / premenopausal females showed three cEC subpopulations based onmedian VEGFR1 and VEGFR2 concentrations: 55.8% cECs had 2,600 VEGFR1 / cell and 2,600 VEGFR2 / cell, the remaining 44.2% cECs had high-VEGFR1 expression of 138,000 VEGFR1 / cell. Among the high-VEGFR1 cECs, half had 39,100 VEGFR2 / cell and the other half had 236,000 VEGFR2 / cell. In some embodiments, the method of treating a human subject for preeclampsia comprises: (a) measuring at least 200,000 VEGFR1 per cEC in cECs isolated from a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, and / or blood pressure monitoring. In some embodiments, the method further comprises measuring at least 10,000 VEGFR2 per cEC.

[0056] In some embodiments, the method of treating a human subject for preeclampsia comprises: (a) measuring at least 200,000 VEGFR1 per circulating CD34+CD31+ cells isolated from a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, blood pressure monitoring, and / or plan for early induction of labor. In some embodiments, the isolated cells are circulating progenitor cells (cPCs). In some embodiments, the cPCs are circulating endothelial cells (cECs).

[0057] Detection of preeclampsia early in pregnancy, e.g., during the first trimester, or before 20 weeks, allows for treatment with prophylactic aspirin therapy (low-dose aspirin). Other treatments for preeclampsia, typically for more severe cases, include administration of anti-hypertensive medications to lower blood pressure, anticonvulsant medication, such as magnesium sulfate, to prevent seizures, and corticosteroids to promote development of the baby's lungs before delivery. In pregnancy, high blood pressure is diagnosed if the systolic pressure is 140 millimeters of mercury (mm Hg) or higher or if the diastolic pressure is 90 mm Hg or higher. In some embodiments, treatment of preeclampsia includes a plan for early induction of labor.

[0058] In some embodiments, the reference amount of surface protein receptors is measured in a healthy control subject. In some embodiments, the subject is a human. In some embodiments, the blood sample comprises 10-20 ml blood. In some embodiments, the blood sample comprises 6-15 ml blood.

[0059] The methods described herein can be applied to early detection of preeclampsia. Obtaining information about the quantity of VEGFRs per cEC in pregnant subjects has diagnostic value at any time up to 34 weeks of gestation and can be predictive when detected before 20 weeks of pregnancy.

[0060] Given that normal high-VEGFR cECs comprising 45% of healthy cECs have 138,000 VEGFR1 / cell and 39,100-236,000 VEGFR2 / cell, and preeclampsia high-VEGFR cECs comprising > 60% of preeclamptic cECs have 150,000 VEGFR1 and 10,000 VEGFR2 per cell, one can easily distinguish cECs associated with preeclampsia by the large increase inhigh-VEGFR cell numbers and VEGFR1 and decrease in VEGFR2. The method can be used to detect both increases and decreases in surface protein receptors.

[0061] As illustrated in the Examples below, a VEGFR1 level above 200 per circulating CD34+CD31+ cell is sufficient to indicate severe preeclampsia with a high degree of sensitivity and specificity. A preferred cutoff value is 236 VEGFR1 per cell, where values above 236 VEGFR1 / cell indicate severe PE, yielding an AUC of 0.76. This cutoff was determined using phycoerythrin-conjugated VEGFR1 antibody R&D Systems) and Quantibrite phycoerythrin calibration beads (BD Biosciences). Those skilled in the art understand that the cutoff value may vary when different VEGFR antibody and / or probes are used or when different fluorophore calibration standards are used.

[0062] In some embodiments, preeclampsia is detected when measured VEGFR1 is above 200 per circulating CD34+CD31+ cell. In some embodiments, preeclampsia is detected when measured VEGFR1 is above 300 per circulating CD34+CD31+ cell. In some embodiments, preeclampsia is detected when measured VEGFR1 is above 400 per circulating CD34+CD31+ cell. In some embodiments, preeclampsia is detected when the measured VEGFR1 per circulating CD34+CD31+ cell is above 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 120,000, 150,000, 180,000, 200,000, 250,000, or 300,000.

[0063] Representative Embodiments

[0064] Embodiment 1. A method of detecting a disorder in a subject, the method comprising: (a) isolating circulating tissue-origin cells from a blood sample obtained from the subject; and (b) measuring the amount of surface protein receptors per circulating tissue-origin cell; wherein a disorder is detected when the amount of surface protein receptors per circulating tissue-origin cell differs from a reference amount or matches with an established threshold of a specific disease.

[0065] Embodiment 2. The method of embodiment 1, wherein the disorder is preeclampsia, the surface protein receptors are vascular endothelial growth factor receptors VEGFR1 and VEGFR2, the circulating tissue-origin cells are circulating endothelial cells (cECs), and wherein preeclampsia is detected when a high-VEGFR cEC population comprises greater than 60% of cECs and exhibits 150,000 VEGFR1 and 10,000 VEGFR2 per cEC.

[0066] Embodiment 3. The method of embodiment 2, wherein the subject is 8-34 weeks pregnant.

[0067] Embodiment 4. The method of embodiment 2, wherein the subject is 8-12 weeks pregnant.

[0068] Embodiment 5. A method of measuring the amount of VEGFRs present in preeclamptic vasculature in a subject, the method comprising: (a) isolating circulating endothelial cells (cECs) from a blood sample obtained from the subject; and (b) measuring the amount of vascular endothelial growth factor receptors (VEGFRs) on the cECs; wherein the amount of VEGFRs relative to the amount of cECs is indicative of the amount of VEGFRs present in placental vasculature of the subject.

[0069] Embodiment 6. The method of embodiment 2 or 5, wherein the cECs are immunopositive for CD34, CD31, and CD146.

[0070] Embodiment 7. The method of embodiment 2 or 5, wherein the isolating comprises immunomagnetic cell capture via contacting the sample with antibody to human CD34.

[0071] Embodiment 8. The method of any of the preceding embodiments, wherein the measuring comprises quantitative flow cytometry.

[0072] Embodiment 9. A method of treating a subject for preeclampsia, the method comprising: (a) performing the method of claim 2 on a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, and / or blood pressure monitoring when the high-VEGFR cEC population comprises greater than 60% of cECs and exhibits 150,000 VEGFR1 and 10,000 VEGFR2 per cEC.

[0073] Embodiment 10. The method of embodiment 1, wherein the reference amount of surface protein receptor is measured in a healthy control subject.

[0074] Embodiment 11. The method of embodiment 1, wherein the surface protein receptor is VEGFR1.

[0075] Embodiment 12. The method of embodiment 11, wherein the population of cECs exhibits 200,000 or more VEGFR1 per cEC.

[0076] Embodiment 13. A method of treating a human subject for preeclampsia, the method comprising: (a) measuring at least 200,000 VEGFR1 per cEC in cECs isolated from a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, and / or blood pressure monitoring.

[0077] Embodiment 14. The method of embodiment 13, further comprising measuring at least 10,000 VEGFR2 per cEC.

[0078] Embodiment 15. The method of any of the preceding embodiments, wherein the subject is a human.

[0079] Embodiment 16. The method of any of the preceding embodiments, wherein the blood sample comprises 10-20 ml blood.

[0080] Embodiment 17. A method of detecting a disorder in a subject comprising one or more steps as described and / or illustrated herein.

[0081] Embodiment 18. A method of detecting preeclampsia in a subject comprising one or more steps as described and / or illustrated herein.

[0082] Embodiment 19. Other methods and compositions as described and claimed herein. EXAMPLES

[0083] The following examples are presented to illustrate the present disclosure and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure.

[0084] Example 1: Identifying age-sex-specific vascular biomarker quantities on circulating vascular cells

[0085] Dysregulated angiogenesis is a characteristic of many vascular diseases and cancers, and better biomarkers of abnormal vessels are needed to evaluate patients and therapies. Two vascular endothelial growth factor receptors, VEGFR1 and VEGFR2, are central regulators of angiogenesis and are biomarkers for many of these diseases. VEGFRs on the cell plasma membrane bind VEGF ligands and initiate downstream angiogenic signaling. The relative abundance of VEGFR1 and VEGFR2 on endothelial cell plasma membranes dictates vascular growth patterns: during sprouting angiogenesis, VEGFR1 is highly expressed by trailing endothelial cells (stalk cells), and VEGFR2 is highly expressed by leading endothelial cells (tip cells)8,10,25and is essential for driving angiogenic sprouting. Further, high VEGFR2 expression has been identified as a biomarker of increased tumor vascular density.47,70

[0086] VEGFR1 is conventionally described as a decoy receptor69because its affinity for VEGF is ten times higher than that of VEGFR2,21,53,75but the resulting angiogenic signaling does not initiate hallmark angiogenic activities, such as endothelial cell proliferation and migration in vitro.78Paradoxically, this decoy role of VEGFR1 does not appear to hold in many pathological conditions, since VEGFR1 overexpression has been correlated with active vascular growth in cancers, ischemic tissue, and obese adipose tissue expansion.13,32,43,62Furthermore, changes in VEGFR protein expression are effective indicators in several clinical prognoses. For instance, immunohistochemical VEGFR1 overexpression is predictive of decreased overall survival in colorectal cancer patients treated by the anti-VEGF agent bevacizumab,80and it can also help identify metastasis inovarian cancer patients.71Despite many efforts to translate VEGFR expression into a reliable indicator for vascular diseases, VEGFR biomarker studies are often qualitative or semiquantitative and thus, are difficult to compare when making VEGFR-driven predictions for personalized prognoses.

[0087] In pursuing quantitative vascular biomarkers, we first need to find ways to reliably detect angiogenic biomarkers and ultimately quantify them. Quantitative flow cytometry has been optimized as a tool for plasma membrane receptor characterization23and applied it to several tyrosine kinase receptors (RTKs), including VEGFRs,34,35Platelet-Derived Growth Factor Receptors,15Epidermal Growth Factor Receptor,17Oxytocin Receptor,15,51,52Axl,22and others.15,17,23RTKs were quantified on several cell types, including healthy35and ischemic33endothelial cells from mouse skeletal muscle, ovarian tumor cell lines,22endothelial cells isolated from breast tumor xenograft,36and patient-derived glioblastoma xenografts.17Collectively, these quantitative studies showed that vascular pathologies, such as ischemic disease and cancers, are associated with dysregulated RTK concentrations, particularly the dysregulation of membranous VEGFRs. However, clinical data acquisition has been impeded by the limited access to human vessel biopsies, which require invasive procedures or expensive imaging modalities (e.g., PET and functional MRI).30It is imperative to identify more accessible angiogenic cells to study vascular biomarkers in human patients.

[0088] Two alternative candidate noninvasive vascular targets, circulating progenitor cells (cPCs) and circulating endothelial cells (cECs), have expanded our knowledge of vascular pathology56,66and have predictive and therapeutic values in various vascular diseases and cancers.5,24,26Both cECs and cPCs closely interact with vascular endothelium during blood vessel formation and remodeling: cECs are shed from vascular endothelium during the expansion or damage of blood vessels,81cPCs promote vessel growth by either merging with or interacting with vascular endothelium,3,46,60and large quantities of VEGFR1+or VEGFR2+cPCs are found in tumor metastases,38,74suggesting that VEGFRs in cPCs are potentially useful as therapeutic biomarkers or therapeutic targets in metastatic cancers. Yet, most cEC and cPC studies have focused on cell enumeration and suggested that increased numbers of cECs6,19or cPCs2–4,20,38,45,67,73,74,83,84are correlated with tumor angiogenesis or vascular diseases.7,85However, cPCs and cECs have been overlooked as accessible indicators of VEGFR expression in the tissue endothelium. This Example presents the use of both cPCs and cECs as reporters of the vascular expression of VEGFRs.

[0089] Developing VEGFR biomarkers starts with establishing baseline levels of plasma membrane VEGFRs in healthy subjects. This Example presents the design of a standardized workflow for isolating cECs and cPCs from blood and characterizing VEGFR1and VEGFR2 localization on plasma membranes. Using this workflow, healthy baselines were established for VEGFR concentrations in cPCs and cECs. Further, because angiogenic capacities are known to vary by age,44, sex,64race,55and menopausal status,49the correlation of these characteristics with VEGFR concentrations on cPCs and cECs was examined. Lastly, the Example demonstrates the ability to differentiate and characterize heterogeneous subpopulations of cECs according to their VEGFR expression levels, specifically VEGFR-high and VEGFR-low. This approach and these measurements are foundational for advancing the VEGFR-driven prognosis of vascular diseases.

[0090] Methods

[0091] Human blood samples

[0092] Human peripheral blood samples were collected from 23 healthy participants (45.6 ± 11.1 years old, 11 females and 12 males). All the females over 50 years old (yo) were menopausal / postmenopausal and the females under 50 yo were peri / premenopausal in this study. None of the participants had been diagnosed with cardiovascular diseases or cancers. None reported hormone replacement therapy history. The blood samples were processed under institutional review board–approved protocols at the BioreclamationIVT facility (now BIOIVT, Westbury, NY). The blood samples were tested following FDA regulations and found negative for HBsAg, HIV 1 / 2 Ab, HCV Ab, HIV-1 RNA, HCV RNA, and STS. The blood samples were stored in K2EDTA, shipped with ice packs on the day of blood draw, and received and analyzed within 24 hours.

[0093] Bulk RBC lysis

[0094] Upon receipt, 9 parts of diluted 1X RBC Lysis Buffer (BioLegend, Cat # 420301) were added to 1 part of whole blood (i.e., 20 mL) and incubated for 15 minutes at room temperature, followed by centrifugation (350 g, 4 °C, for 5 min). Lysed whole blood cells were washed twice with 10 mL stain buffer (PBS supplemented with 0.5% bovine serum albumin, 0.09% sodium azide, and 2 mM EDTA), centrifuged (350 g, 4 °C, for 5 min), and resuspended in 1 mL stain buffer for the subsequent immunomagnetic enrichment.

[0095] CD34+ cell enrichment

[0096] To every 0.5 mL of lysed RBC cell suspension (approx.2 × 107PBMCs from 10 mL whole blood54), 25 μL of DSB-CD34 biotinylated antibody was added. The DSB-CD34 biotinylated antibody (0.4 mg / mL) was prepared by conjugating purified human CD34 antibody (clone My10 or 581, BioLegend) to DSB-X biotin (DSB-X Biotin Protein Labeling Kit, Cat No. D-20655) per the manufacturer’s protocol, and according to our prior work.33,34,36The mixture was gently pipetted to mix and then incubated on ice for 20 min. After a washingstep in 2 mL of ice-cold stain buffer, it was centrifuged at 350 g at 4 °C for 10 min without braking. The cell pellets were resuspended in 1 mL stain buffer and transferred to a 1.5-mL Eppendorf tube.

[0097] Next, 75 μL FlowComp Dynabeads coated with streptavidin (Invitrogen, cat no. 11061D) were vortexed thoroughly (approx.10 seconds), washed twice with 1 mL stain buffer, and resuspended in 75 μL of ice-cold stain buffer. Thereafter, 75 μL bead solution was added to the 1 mL DSB-CD34 labeled cell suspension. The bead-cell mixture was incubated on a rotator (approx.6 rpm) at 4 °C for 20 min. Next, the bead-cell mixture was diluted at a 1:1 ratio with stain buffer and placed in a magnetic separator (DynaMag-5) for 2 min. After removal of the bead-free, unbound CD34-negative cell suspension by careful pipetting, CD34+ cells remained on the wall of the tube due to the magnetic field. The tubes were then removed from the magnetic separator so that beads were released from the magnet walls. The bead-cell mixture was collected, resuspended in 1 mL stain buffer, and placed back into the magnetic separator for at least two more washing steps, after which all the beads were combined in a new Eppendorf tube with 1 mL of biotin-rich release buffer (FlowComp) and incubated on the rotator at 4 °C for 10 min. By pipetting the cell suspension 10 to 15 times, the CD34+ cells were detached from the beads by biotin-streptavidin competition. Then the tube was again placed in the magnetic separator. After 2 minutes, bead-free CD34+ cells in the supernatant were carefully collected and transferred to a 5-mL polystyrene FACS tube (BD Biosciences, New Jersey). Cells were concentrated via centrifugation at 400 g for 5 min at 4 °C. Cell-free supernatant was decanted. Lastly, the CD34+ cells were resuspended in 200–400 μL stain buffer and kept on ice until flow cytometry immunostaining.

[0098] Immunostaining and flow cytometric acquisition of CD34+ cells

[0099] Enriched CD34+ cells were aliquoted into 20 μL / FACS tubes and stained with APC- conjugated CD146 and APC-Cy7-conjugated CD31 antibodies per the manufacturer’s recommendation (5 μL / test, BioLegend). Either phycoerythrin-conjugated VEGFR1 (R&D Systems) or phycoerythrin -conjugated VEGFR2 (BioLegend) was added to each test tube at the saturating concentration (14 μg / mL).34Samples were incubated in the dark for 40 minutes at 4 °C, washed twice with 2 mL of stain buffer, centrifuged at 400 g at 4 °C for 5 min, and resuspended in 100 μl of stain buffer. To assess cell integrity, 2 μl of Sytox Blue was finally added to each tube as a liquid drop-in. Corresponding fluorescence-minus-one (FMO) controls were used for evaluation of the nonspecific binding of monoclonal antibodies to identify the positive / negative boundary for each fluorophore signal.96The FMO control contained all the fluorophores in a panel, except for the one being measured.

[0100] Flow cytometry was performed on either of two instruments: an LSR Fortessa (BD) or a Beckman Coulter CytoFLEX S Flow Cytometer. Samples were vortexed immediately prior to placement in the flow cytometer. Prior to sample acquisition, phycoerythrin voltage settings were finalized and Quantibrite PE (phycoerythrin) beads (BD, cat. no.340495) were collected. Flow cytometric data analysis was performed using FlowJo analytical software or Kaluza analytical software.

[0101] The phycoerythrin fluorophore offers several advantages, such as its high photostability, pH independence, and small size.12,28In particular, due to its 1:1 protein / fluorophore ratio, the number of phycoerythrin molecules per cell equals the number of phycoerythrin -conjugated receptors per cell; applying qFlow with phycoerythrin - conjugated VEGFR antibodies allows absolute quantification of membrane VEGFR levels. The precision and accuracy of quantitative flow cytometry (qFlow) profiling have been rigorously tested.9,15,18,34,36,79,82

[0102] Quantitative flow cytometric data analysis

[0103] The levels of VEGFR per cell were acquired by converting the PE fluorescence intensity to the number of phycoerythrin molecules per cell, using Quantibrite PE (phycoerythrin) beads as previously described.15,18,33,34,36Quantibrite PE beads are polystyrene beads conjugated with different densities of phycoerythrin molecules: low (474 phycoerythrin molecules / bead), medium-low (5,359 phycoerythrin molecules / bead), medium-high (23,843 phycoerythrin molecules / bead), and high (62,336 phycoerythrin molecules / bead). The geometric mean phycoerythrin values of the respective bead subsets were exported and used to calculate m (slope) and b (intercept) in linear regression(Equation 1). Because phycoerythrin molecules are conjugated with anti-VEGFRs at a 1:1ratio, this equation can convert the phycoerythrin readout of cells directly to the number of phycoerythrin-receptors / cell. A calibration curve was established to translate the phycoerythrin fluorescence level to the phycoerythrin quantity.

[0104] 1

[0105] Phycoerythrin (PE in Equation 1) fluorescence intensities were recorded for individual CD34+ cells as geometric mean values, which were exported to Excel files. False- positive phycoerythrin signals, commonly due to cell auto-fluorescence, were collected from PE-FMO samples and denoted as phycoerythrin background fluorescence. In order to accurately quantify phycoerythrin -receptor levels, the phycoerythrin backgroundfluorescence was subtracted from the phycoerythrin fluorescence of phycoerythrin -stained samples, using a weighted integral approach (Equation 2).

[0106] Equation 2

[0107] PEabsoluteis the number of receptors per cell obtained after subtracting PEbackground,which was obtained from PE-FMO samples. PEstained is the unsubtracted receptor levelmeasured in a phycoerythrin -stained sample. Nstainedand nPE-FMOare the cell numbers collected from phycoerythrin -stained and phycoerythrin -FMO samples, respectively.

[0108] Pooling samples

[0109] The lengths of the datasets were set equal to ensure individual datasets contributed equally to the pool. The length of each cPC dataset was set to 10,000 and the length of each cEC dataset was set to 1000. This was done by using the “datasample” function in Matlab. This function did not change the median or IQR values of the VEGFR distributions of each dataset. The equal-sized datasets of peri / premenopausal females, menopausal / postmenopausal females, males above 50 years old (yo), and males below 50 yo were separately pooled and analyzed using Matlab.

[0110] Mixture modeling analysis

[0111] A previously established Gaussian mixture modeling17was applied to identify log- normal subpopulations within each cEC distribution, described by its median and IQR. A detailed description of heterogeneity quantification via this method was provided by S. Chen et al.17Ideally, the optimal number of cell subpopulations is determined by the Bayesian information criterion (BIC) values, where the lowest value indicates the best fit.17,77However, when the number of data points is low, the conventional BIC standard can result in overfitting. Therefore, in this study, instead of relying on the BIC standard, the mixture modeling algorithm was set to identify three possible subpopulations, which were denoted as low-, intermediate-, and high-VEGFR cEC subpopulations. Although there could be more than three cEC subpopulations, our fitting results provided a realistic estimate of the major cEC subpopulations in healthy blood samples.

[0112] Statistical analysis

[0113] The distributions of VEGFR levels (median and IQR) on CD34+CD31+ cells and CD34+CD31+CD146+ cells were checked using the Shapiro-Wilk test. For variables that are not normally distributed but are right-skewed with skewness >1.5, log transformation was applied. One-way ANOVA or the two-sample t-test were first used to assess the relationship between VEGFR levels and the three independent factors (sex, race, and age category). Factorial ANOVA was then performed and included the three independent factors and their possible two-way interactions. The predicted least-squares means of the VEGFR levels were obtained at different levels of a factor that was adjusted for other factors in the model. For significant two-way interactions, the least-squares means of a factor for each level of the other factor were obtained with a 95% confidence interval (CI). Significant least- squares means comparison with 95% CI for age by sex groups were also reported. All the statistical tests were two-sided at a significance level of 0.05, and statistical analyses were performed using SAS 9.4 (SAS Inc., Cary, NC). A Cohen’s d (a standardized effect size) was estimated from the factorial ANOVA. As a general guide, Cohen’s d values of 0.3, 0.5, 0.8, 1.2, and 2 correspond to mild, moderate, large, very large, and huge effect sizes, respectively.50 Effect sizes were used to quantitatively compare the results of studies done in a different setting since Cohen’s d is independent of sample size.

[0114] Ethics approval

[0115] The human blood samples were collected under Institutional Review Board– approved protocols at the BioreclamationIVT (now renamed as BIOIVT) facility (Westbury, NY).

[0116] Results

[0117] Blood-based proteomic analysis workflow establishes VEGFR quantities on circulating vascular cells

[0118] The four-step workflow was designed as follows: (1) cPC / cEC enrichment and stratification, (2) plasma membrane VEGFR quantification, (3) statistical analysis of the correlation between subject characteristics (such as age, sex, race, and menopausal status) and inter-sample variations, and (4) mixture modeling analysis for dissecting intra-sample heterogeneity.

[0119] Blood samples were obtained from 23 healthy subjects (6 menopausal / postmenopausal and 5 pre / peri-menopausal females and 12 age-matched males). Subject characteristics such as age, sex, and race are listed in Table 1. Subjects’ ages ranged from 25 to 60 yo, with a median of 51 yo and an interquartile range (IQR) of 18 yo (37 –55 yo). The median and IQR are reported here due to the nonnormal age distribution of the samples (Shapiro-Wilk test p = 0.0156). The subjects were Black (non-Hispanic)(52.2%, n = 12), white (non-Hispanic) (26.1%, n = 6), and Hispanic (21.7%, n = 5). Since human research studies are often limited by sample sizes, we focused on large effect sizes (Cohen’s d >1.2) 65 and 95% confidence intervals when considering conclusions drawn from statistical tests.

[0120] Table 1. Healthy patients’ demographic information and descriptive data (median and IQR, %cECs)..

[0121] cECs constitute 0.0001–0.01% of peripheral blood mononuclear cells;40however, flow cytometric detection requires the targeted cell frequency to be above 0.01%.72Therefore, CD34+ immunomagnetic isolation36was included to enrich cPCs from the peripheral blood and increase cEC frequency for the subsequent VEGFR quantification. To exclude dead cells and platelets, we first selected (gated) large cells, using 6 μm phycoerythrin (PE) calibration beads as a size reference (Fig.1A). Debris and aggregates were excluded to reduce noise from the signal (Fig.1B). Our results confirmed prior work showing that CD34+ cPCs also express CD31 (Fig.1C).39,59,63,68Finally, we separated cECs from cPCs with an endothelial-specific marker, CD146.42The enhanced cEC frequency was 0.98 ± 0.29% (mean ± SEM, n = 23) in the CD34-enriched flow samples (Fig.1D).

[0122] Subsequent data analysis focused on three goals: (1) using frequency histograms to establish plasma membrane VEGFR concentrations on single cECs and cPCs, (2) using factorial ANOVA and effect size (Cohen’s d) to identify correlations between subject characteristics (sex, age, race, and menopausal status) and the respective median concentrations of VEGFRs on cECs and cPCs, and (3) adapting a mixture modeling approach, established for quantitative flow cytometry 79 to characterize VEGFR heterogeneity in cECs.

[0123] cPCs and cECs exhibited heterogeneous plasma membrane expression of VEGFRs

[0124] The numbers of VEGFR1 and VEGFR2 molecules on the plasma membranes of cECs and cPCs were quantified in a cell-by-cell manner and plotted them on log-scale histograms as previously described.16,79(Fig.2A–B, 2D–E) Median VEGFR concentrations and interquartile ranges (IQRs) of these distributions were calculated and reported, along with subject age, sex, and race, in Table 2. It should be noted that the threshold for specific binding of human VEGFR antibodies to the cell membrane is 200–500 VEGFRs / cell on average.18Therefore, VEGFR1 plasma membrane expression on cPCs was detected in only the 30% of the subjects who were (post)menopausal females, and VEGFR2 plasma membrane expression on cPCs was not detected. On cECs, VEGFR1 and VEGFR2 plasma membrane expression was detected in all subjects.

[0125] The varied plasma membrane VEGFR concentrations across the individuals were quantified and reported as the median of VEGFR distributions: VEGFR concentrations ranged from 10 to 4700 VEGFR1s per cPC (Fig.2C), 10 to 400 VEGFR2s per cPC (Fig. 2C), 900 to 124,000 VEGFR1s per cEC (Fig.2F), and 1200 to 182,000 VEGFR2s per cEC (Fig.2F). Together, these data illustrate the inter-subject variability of VEGFR concentrations on cECs and cPCs across healthy individuals.

[0126] VEGFR plasma membrane localization in healthy subjects is associated with age-sex interaction effects

[0127] Age,44sex,64race,55and menopausal status49,58can alter angiogenic capacity by shifting the balance between anti- and pro-angiogenic factors, influencing individual susceptibility to vascular diseases and cancers. We examined whether these characteristics correlated with intersample VEGFR variations in cPCs and cECs. All the females in this study aged above 50 years old (yo) self-reported as (post)menopausal, and those below 50 yo were self-reported as peri / premenopausal; menstrual status was thus considered an age- sex interaction effect. We used the age of 50 yo as a cutoff for the statistical interpretation of the effects of age on median VEGFR concentrations of cPCs and cECs from the healthy females and those from age-matched males.

[0128] Menstrual status was significantly correlated with cPC and cEC VEGFR concentrations. We detected six statistically significant sex-by-age interactions (p < 0.05), as summarized in Table 2. The median VEGFR1 concentrations of cPCs were higher in menopausal / postmenopausal females (n = 6) than in peri / premenopausal females (n = 5) (p = 0.0014) and age-matched males (p = 0.0054). The median VEGFR1 concentrations of cECs were higher in peri / premenopausal females than in menopausal / postmenopausal females (p = 0.002), age-matched males (28–44 yo, p = 0.008), and older males (52–57 yo, p = 0.0082). Moreover, median VEGFR2 concentrations of cECs were higher in peri / premenopausal females than in menopausal / postmenopausal females (p = 0.0265). None of the three factors (age, sex, and race) were independently associated with VEGFR concentrations. No significant sex-by-race or race-by-age interaction was detected in VEGFR concentrations.

[0129] Table 2. Healthy patients’ demographic information and descriptive data (median and IQR, %cECs). Significant sex*age group comparisons on VEGFR concentrations of cECs and cPCs based on factorial ANOVA. Median values of VEGFR distributions were compared across 23 healthy samples. Six significant comparisons were identified as p-value < 0.05 with a 95% confidence interval. A Cohen’s D (a standardized effect size) was estimated from the factorial ANOVA. The present study yielded very large Cohen’s D values, ranging between 1.63 and 2.56, indicating that the two groups’ means differed by around 2 standard deviations.

[0130] To assess the magnitude of the differences between peri / premenopausal females, menopausal / postmenopausal females, and males, we computed effect sizes (Cohen’s d) independent of the sample size. As a general guide, effect sizes of 0.3, 0.5, 0.8, 1.2, and 2 correspond to mild, moderate, large, very large, and huge effect sizes, respectively.65 A large effect size is desired in biomarker research because a biomarker is useful when biomarker levels of two study groups are far apart. In the present study, the effect sizes were “very large”, ranging between 1.63 and 2.56, indicating that the two groups’ means differed by 1.63 to 2.56 standard deviations (Table 3). These large effect sizes indicate that inter- sample variations of VEGFR plasma membrane localization in healthy subjects are significantly associated with age-sex interaction effects, like menstrual status, and are considerations when studying VEGFR membrane localization and signaling mechanisms.

[0131] Table 3. Descriptive statistics of VEGFR expression baselines in each sex*age group. Blood samples are pooled based on sex*age interaction categories. Median and IQR values of VEGFR concentrations are extracted from the pooled cell-by-cell VEGFRdistributions to represent the healthy levels of VEGFR1 and VEGFR2 on cECs and cPCs in these sex*age groups. * Indicates the values are below the previously established quantifiable threshold (median 500 VEGFRs / cell) [S. Chen 2017, book chapter]. Values of VEGFR2 per cPC are not shown because they were all below the 500 VEGFR / cell threshold.

[0132] VEGFR1 plasma membrane localization in cPCs is detected only in menopausal / postmenopausal females

[0133] Increasing evidence has shown that menopause alters angiogenic capacities, as manifested by decreased blood VEGF concentrations, decreased endothelial cell proliferation,58and increased risks for cardiovascular diseases.44Here, the data illustrate that, though the differences in VEGFR2 levels in cPCs were negligible between samples, cPC VEGFR1 distributions were increased (more right-skewed median levels) in menopausal / postmenopausal females compared with those in peri / premenopausal females (Fig.3A). VEGFR1 plasma membrane localization in cPCs was thus expected to play a more significant role in regulating angiogenesis in menopausal / postmenopausal females than in peri / premenopausal females. Therefore, to isolate the nonpathological variable of menopause, we established separate VEGFR baselines of cPCs for menopausal / postmenopausal females and peri / premenopausal females (Fig.3B).

[0134] A median of 650 VEGFR1s / cPC were detected on the plasma membrane in the pooled menopausal / postmenopausal females’ cPCs, whereas the pooled peri / premenopausal females’ cPCs had a median of only 88 VEGFR1s / cPC (Fig.3B). Membranous VEGFR concentrations below 500 VEGFRs / cell are typically described as little-to-no, because prior tests of nonspecific antibody binding results in the quantification of between 200-500 receptors.18Male cPCs also exhibited low-to-no plasma membrane VEGFR1s (Table 3).21Overall, the numbers of VEGFR1 molecules localized to the membrane on cPCs in peri / premenopausal females and males (88–330 VEGFR1s / cPC) were considered negligible. VEGFR2 plasma membrane expression on cPCs was also negligible in all healthy blood samples (median, <200 VEGFR2s / cPC).

[0135] High-VEGFR cEC subpopulations are detected in peri / premenopausal females and males of all ages

[0136] cECs shed from various tissues exhibit considerable vascular heterogeneity (e.g., different gene expressions, functions, and morphology).41Identifying the normal cEC subpopulations in healthy individuals is helpful to distinguishing abnormal cEC subpopulations shed from diseased vascular beds. To this end, we investigated VEGFR- based cEC subpopulations in healthy individuals, taking into account the significant age-sex interactions. (Table 2).

[0137] cECs in peri / premenopausal females consisted of two VEGFR populations: VEGFR- low and VEGFR-high, while the menopausal / postmenopausal group only possessed the VEGFR-low cEC population (Fig.4). The VEGFR-low and VEGFR-high cEC subpopulations were identified and quantified by the median VEGFR concentrations: (1) cECs in menopausal / postmenopausal females were generally VEGFR-low, presenting ~3000 VEGFR1s and ~3000 VEGFR2s per cell (Fig.4A and 4B); (2) about half of the cEC population in the peri / premenopausal female group presented one-to-two orders of magnitude more VEGFRs, having 138,000 VEGFR1s / cell and 39,000 to 236,000 VEGFR2s / cell (Fig.4C and 4D); and (3) a quarter of the cEC population in males presented high VEGFR numbers, having 206,000 VEGFR1s / cell and 155,000 VEGFR2s / cell (Fig.4E and 4F). Table 3 summarizes the descriptive statistics of VEGFR concentrations in cPCs and cECs, including the medians and IQRs, for females above 50 years old and under 50 years old and for age-matched male groups.

[0138] Discussion

[0139] Sex differences in endothelial cells and angiogenic regulators are known to contribute to sex-specific mechanisms in angiogenesis.64The noninvasive, vascular cell-targeted biomarker approach described herein has revealed significant, quantifiable differences inVEGFR1 and VEGFR2 plasma membrane localization in cECs and cPCs between menopausal / postmenopausal females, peri / premenopausal females, and males of all ages. Our findings, for the first time, provide quantitative insights into how sex-age interactions influence VEGFR plasma membrane localization in circulating angiogenic cells. Separate healthy baselines for VEGFR expression on the plasma membranes of cECs and cPCs were established for menopausal / postmenopausal females, peri / premenopausal females, and males of all ages. This presents a first step towards precision medicine and supports future investigations into the predictive values of circulating vascular cells in VEGFR-driven prognoses.

[0140] Different cEC subpopulations may be attributed to aging-dependent sex differences in angiogenesis. Notably, menopausal / postmenopausal females’ cECs homogeneously exhibit low plasma membrane VEGFR levels, whereas peri / premenopausal females’ cECs are more heterogeneous, consisting of ~50% VEGFR-low and 50% VEGFR-high cECs.26(Fig.4 A–D) Males possess a smaller fraction (27%) of VEGFR-high cEC subpopulations, which may originate from male-prone angiogenic processes, such as visceral fat expansion– induced adipose angiogenesis.57Together, these data suggest distinguishable membranous VEGFR distribution patterns in cEC subpopulations of different sex-age groups (Fig.5).

[0141] One possible source of the VEGFR-high cEC subpopulations in peri / premenopausal females is the turnover of vascular endothelial cells due to estrogen-enhanced angiogenesis during menstruation.48During the menstrual cycle, angiogenesis forms new endometrial endothelium;50,61the high-VEGFR cECs are potential non-invasive proxies for studying endometrial angiogenesis.62Our non-invasive method has the potential to detect the upregulation of VEGFR2 in endometriotic vessels11by quantifying the VEGFR2 molecules on cECs obtained from women with endometriosis. In addition, compared to soluble biomarkers such as DNA, mRNA, metabolites, and soluble proteins (such as VEGF and soluble VEGFR), our cell-based VEGFR measurements offer a more direct representation of endothelial cell phenotypes in the tissue origin. Thus, measuring the VEGFRs on the membrane is more pertinent to endometriotic angiogenesis than measuring soluble VEGFRs, which did not show a correlation with the occurrence of endometriosis.76

[0142] Future studies using single-cell RNA sequencing can confirm cEC origins: this has been performed successfully on vascular cells of brain,29,37lung,29,37heart,37and endometrium.41Although the origins of the VEGFR-high cECs in healthy males and peri / premenopausal females need to be further identified, cECs shed from diseased vasculature and cancers are expected to outnumber their normal counterparts (Table 4), allowing for precise identification of diseased cEC subpopulations via mixture modeling.

[0143] Table 4. Reported cEC counts in disease studies of vascular disorders and cancers. cEC counts are described by either median (range) or mean ± SD, depending on the format in the original reports.1. Wang, C., et al. Clin. Hemorheol. Microcirc. (2005). 2. Lee, K. W., et al. Blood (2005) doi:10.1182 / blood-2004-03-1106.3. Makin, A. J., et al. Eur. Heart J. (2004) doi:10.1016 / j.ehj.2003.04.001. 4. Bull, T. M. et al. C Thromb. Haemost. (2003) doi:10.1160 / th03-04-0251. 5. Smadja, D. M. et al. Circulation (2009) doi:10.1161 / CIRCULATIONAHA.108.808246. 6. Nadar, S. K., et al. Thromb. Haemost. (2005) doi:10.1160 / TH04-12-0795. 7. Farinacci, M. et al. Res. Pract. Thromb. Haemost. (2019) doi:10.1002 / rth2.12158. 8. Alessio, A. M. et al. Int. J. Med. Sci. (2013) doi:10.7150 / ijms.6887. 9. Nizzoli, M. E. et al. Am. J. Hematol.95, E187–E188 (2020). 10. Guervilly, C. et al. J. Infect. Dis. (2020) doi:10.1093 / infdis / jiaa528. 11. Beerepoot, L. V. et al. Ann. Oncol.15, 139–145 (2004). 12. Vaz Salgado, M. Á. et al. J. Clin. Oncol. (2019) doi:10.1200 / jco.2019.37.15_suppl.e13517. 13. Bidard, F. C. et al. Ann. Oncol. (2010) doi:10.1093 / annonc / mdq052. 14. Rahbari, N. N. et al. Prognostic value of circulating endothelial cells in metastatic colorectal cancer Study population.8, 37491–37501 (2017).

[0144] cPCs play a role in vascular regeneration, and the distinct VEGFR1 membrane localization on menopausal / postmenopausal cPCs suggests a menopause-specific angiogenic mechanism. This is further supported by the fact that VEGFR1 membrane localization is undetectable in peri / premenopausal females and males of all ages. Menopause is a major sex difference that depends on age and correlates with increased risk for vascular diseases and cancers,27so presence of VEGFR1+cPCs in menopausal / postmenopausal females begs the question of its implication on cancer risk. We propose further studies of the menopausal / postmenopausal cPC connection to cancer risk for three reasons: (1) VEGFR1+cPCs are linked to tumor vascularization and metastasis,30(2) VEGFR1 signaling on cPCs promotes cPC recruitment to activated or tumor blood vessels, giving rise to endothelial cells or myeloid cells that produce essential vascular growth factors like VEGF-A,1,45and (3) VEGFR1+cPC clusters make up pre-metastatic sites, which permit the attachment of circulating tumor cells and endothelial progenitor cells to form metastases.38Further studies are needed to determine whether the increased VEGFR1 plasma membrane localization in cPCs is associated with the higher risk of vascular diseases and cancer seen in menopausal / postmenopausal females.14,31

[0145] Both cECs and cPCs are accessible proxies that provide quantitative molecular insights into VEGFR plasma membrane localization and signaling in hosts’ blood vessels. This work provides a standardized, non-invasive method and baseline data for future studies to quantitatively profile VEGFR plasma membrane localization in cECs and cPCs from patients with angiogenic disorders (e.g., cancers, obesity, and cardiovascular diseases). Utilizing our findings as a baseline, future studies may show that VEGFRs on cECs and cPCs are predictive biomarkers for stratifying patients with vascular dysfunction for more effective vascular-targeted strategies. The present findings emphasize the considerations ofincorporating the biology of sex and age differences into paradigms for angiogenesis research. This approach enables accessible, quantitative, and standardizable protein biomarker analyses that are much needed to advance predictive vascular biomarker development.

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[0232] Example 2: Cell-to-cell VEGFR distributions in cECs of a pregnant woman at her antenatal visit

[0233] Cell-to-cell VEGFR distributions in cECs of a pregnant woman at her antenatal visit are shown in FIG.7. The blood test described in Example 1 was conducted on a pregnant woman (unknown diagnosis) using a sample of 10 mL whole blood to demonstrate the technical and statistical capabilities of the test. The test distinguished between a low- VEGFR1 cEC population expressing 2647 VEGFR1 / cell and a high-VEGFR1 cEC population expressing 8000 VEGFR1 / cell. The low-VEGFR2 population expressing 960 VEGFR2 / cell and the high-VEGFR2 population expressing 17,000 VEGFR2 / cell. The grey- shaped plot represents raw data before applying mixture modeling analysis.

[0234] Example 3: Confirmation and Validation of Test for Preeclampsia

[0235] The blood test protocol was performed as illustrated in FIG.8A, by lysing RBCs from a sample of whole blood and isolating CD34+ cells. These CD34+ cells were labeled with fluorophore antibodies, including CD31 and VEGFR antibodies, and the fluorophores were measured using flow cytometry. These measurements were then used to determine biomarker abundance. The protocol can be carried out in about 4 hours, with about 1.5 hours of that being hands-on.

[0236] The targeted cell type is present at 900 to 30,000 cells / mL of blood in pregnant women (mean: 10,000 cells / mL). To verify the test's accuracy in measuring biomarker levels on these cells, blood samples were spiked with 800 to 80,000 cells / mL of cultured cells. Cells were retrieved using the above protocol, and their biomarker levels were compared tocells directly from the culture flask. The protocol retrieves the targeted cells, even at low abundance (800 cells / mL), and measures biomarker levels without altering biomarker expression.

[0237] As shown in FIG.8B, the protocol does not alter biomarker level on the cell standard. The upper panel shows cell that went through the protocol, and the lower panel shows cells that had not gone through the protocol.

[0238] FIGS.9A and 9B show that the test results are reproducible. FIG.9A shows the results of a test performed on a sample obtained at Patient 012’s first visit, at 36 weeks, 5 days, which showed 140 VEGFR1 / cell and 1400 VEGFR2 / cell. FIG.9B shows the results of a test performed on the same patient’s second visit, at 39 weeks, 3 days. This second visit’s test result showed 200 VEGFR1 / cell and 1100 VEGFR2 / cell. This VEGFR1 test shows higher sensitivity than the conventional urine protein to creatinine ratio test, which is one of the PE diagnostic markers. For preeclampsia diagnosis, the American College of Obstetricians and Gynecologists and other guidelines define diagnostic criteria that include blood pressure elevation and proteinuria. Blood pressure elevation is greater than or equal to 140 / 90 mmHg on two occasions after 20 weeks of gestation. Proteinuria is defined as greater than or equal to 300 mg / 24 hours of protein in urine, or a urine protein to creatinine (P:C) ratio of greater than or equal to 0.3 mg / mg. Both urine P:C ratio and VEGFR1 were measured in the patients. As shown in FIGS.10A-10B, the P:C ratio test exhibited 63% sensitivity at detecting control versus severe PE (10A), while the VEGFR1 test exhibited 91% sensitivity (10B).

[0239] Example 4: Clinical Data Analysis of Preeclampsia Test

[0240] The blood test protocol was again performed as illustrated in FIG.8A, by lysing RBCs from a sample of whole blood and isolating CD34+ cells. These CD34+ cells were labeled with fluorophore antibodies and the fluorophores were measured using flow cytometry. These measurements were then used to determine biomarker abundance. The protocol can be carried out in about 4 hours, with about 1.5 hours of that being hands-on.

[0241] For this study, 37 pregnant women were recruited. The patients were categorized into three groups based on their clinical diagnoses: control, mild preeclampsia (PE), and severe PE. Patient characteristics are shown in Table 5. Fresh whole blood samples were tested within 30 hours of collection.

[0242] Table 5. Participant Characteristics

[0243] The blood test outcomes include six measurements, among which the geometric mean (GM) values of VEGFR1 concentration per circulating progenitor cells (CPCs) demonstrate statistical significance in distinguishing between control and any PE cases, and also between control and severe PE cases.

[0244] Table 6a. Values of clinical labs overall and by type of PE; median (IQR)

[0245] Table 6b. Values of clinical labs overall and by type of PE, twins excluded; median (IQR)

[0246] Figures 11A and 11B are scatterplots that show VEGFR1 concentration for each patient as a single data point. Twins (2) were excluded from the data. In FIG.11A, superimposed PE, patients who had hypertension before week 20 (SIPE) are shown separately. In FIG.11B, PE and SIPE are combined.

[0247] The diagnostic performance of the VEGFR1-based test achieved an area under the curve (AUC) of 0.78 (95% CI), with 78% sensitivity at 75% specificity for identifying severe PE. The optimal cutoff value is 236 VEGFR1 per cell, where values above 236 VEGFR1 / cell indicate severe PE, yielding an AUC of 0.76. These results are plotted in FIG.12 and performance at the optimal cutpoint is shown in Table 7.

[0248] Table 7. Performance of VEGFR1 at the optimal cut pointTwins and Post artum PE excluded

[0249] Example 5: BMI moderately correlates with VEGFR1 concentrations

[0250] Body mass index (BMI) was evaluated in subjects from Example 4 above. As shown in Table 8, BMI correlates moderatly with VEGFR1 concentrations.

[0251] Table 8. Correlations between VEGFR1 and clinical characteristics

[0252] Combining BMI and VEGFR1 data achieved an AUC of 0.85 (95% CI, 0.68-1), with 89% sensitivity and 75% specificity for identifying severe PE. This was not, however, statistically different from evaluating VEGR1 alone (p=0.12).

[0253] Table 9a. Comparison between VEGFR1 and BMI+VEGFR1 for control versus any PE

[0254] Table 9b. Comparison between vEGFR1 and BMI+VEGFR1 for control versus severe PE

[0255] Most patients with obesity had severe PE. The test identified 83% severe PE cases in patients without obesity (5 out of 6). These data are shown in FIG.13A. FIG.13B shows the sensitivity and specificity achieved with VEGFR1 alone and with VEGFR1+BMI.

[0256] Example 6: VEGFR2 is not effective for testing preeclampsia

[0257] VEGFR2 has traditionally been regarded as a signaling protein in angiogenesis. The present evaluation shows that VEGFR2 is not effective for testing for preeclampsia. FIG.14 plots the number of VEGFR2 per cell for control, mild PE, severe PE, and postpartum PE. Twins (2) were excluded. PE and SIPE were combined. Table 10a shows the comparison between VEGFR measures for control versus any PE case, and Table 10b shows the comparison between VEGFR measures and control versus severe PE.

[0258] Table 10a. Comparison between VEGFR measures for control versus any PE,

[0259] Table 10b. Comparison between VEGFR measures for control versus severe PE

[0260] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this disclosure pertains.

[0261] From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the disclosure is not limited except as by the appended claims.

Claims

What is claimed is:

1. A method of detecting a disorder in a subject, the method comprising: (a) isolating circulating tissue-origin cells from a blood sample obtained from the subject; and (b) measuring the amount of surface protein receptors per circulating tissue-origin cell; wherein a disorder is detected when the amount of surface protein receptors per circulating tissue-origin cell differs from a reference amount or matches with an established threshold of a specific disease.

2. The method of claim 1, wherein the isolated cells are immunopositive for CD34 and CD31.

3. The method of claim 2, wherein the surface protein receptors are VEGFR1, and the disorder is preeclampsia.

4. The method of claim 3, wherein preeclampsia is detected when greater than 200 VEGFR1 per isolated cell are measured.

5. The method of claim 4, wherein the measured VEGFR1 per isolated cell is a geometric mean of VEGFR1 per isolated cell.

6. The method of any one of claims 2 to 5, wherein the subject is 8-34 weeks pregnant.

7. The method of claim 6, wherein the subject is 8-12 weeks pregnant.

8. A method of measuring the amount of VEGFRs present in preeclamptic vasculature in a subject, the method comprising: (a) isolating circulating endothelial cells (cECs) from a blood sample obtained from the subject; and (b) measuring the amount of vascular endothelial growth factor receptors (VEGFRs) on the cECs; wherein the amount of VEGFRs relative to the amount of cECs is indicative of the amount of VEGFRs present in preeclamptic vasculature of the subject.

9. The method of claim 8, wherein the cECs are immunopositive for CD34 and CD31, and, optionally, CD146.

10. The method of claim 5 or 8, wherein the isolating comprises immunomagnetic cell capture via contacting the sample with antibody to human CD34.

11. The method of any of the preceding claims, wherein the measuring comprises quantitative flow cytometry.

12. A method of treating a subject for preeclampsia, the method comprising: (a) performing the method of claim 4 on a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, blood pressure monitoring, and / or plan for early induction of labor when more than 200 VEGFR1 per isolated cell are measured.

13. The method of claim 1, wherein the reference amount of surface protein receptor is measured in a healthy control subject.

14. The method of claim 1, wherein the surface protein receptor is VEGFR1.

15. The method of claim 14, wherein the population of cECs exhibits more than 200 VEGFR1 per cEC.

16. A method of treating a human subject for preeclampsia, the method comprising: (a) measuring more than 200 VEGFR1 per circulating CD34+CD31+ cells isolated from a blood sample obtained from the subject; and (b) treating the subject with low dose aspirin, bedrest, blood pressure monitoring, and / or plan for early induction of labor.

17. The method of any of the preceding claims, wherein the isolated cells are circulating progenitor cells (cPCs).

18. The method of claim 17, wherein the cPCs are circulating endothelial cells (cECs).

19. The method of any of the preceding claims, wherein the subject is a human.

20. The method of any of the preceding claims, wherein the blood sample comprises 10- 20 ml blood.

21. The method of any of claims 1 to 19, wherein the blood sample comprises 6-15 ml blood.

22. A method of detecting a disorder in a subject comprising one or more steps as described and / or illustrated herein.

23. A method of detecting preeclampsia in a subject comprising one or more steps as described and / or illustrated herein.

24. Other methods and compositions as described and claimed herein.

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