Sampling and testing process for brain health and injury from various sources and correlations therebetween

Abstract

A process for determining an extent of a central nervous system (CNS) specific neurological condition in a subject. The process including collecting a biological sample of a biofluid other than a venous or arterial blood draw from the subject into a collection device; measuring a quantity of first biofluid biomarker, a breakdown product (BDP) thereof, or a combination thereof from the sample; and correlating the quantity of first biofluid biomarker, BDP, or combination thereof with a quantity of first biofluid biomarker, BDP, or combination thereof from the venous or arterial blood draw from the subject to determine the extent of the CNS specific neurological condition in the subject.

Description

SAMPLING AND TESTING PROCESS FOR BRAIN HEALTH AND INJURY FROM VARIOUS SOURCES AND CORRELATIONS THEREBETWEENGOVERNMENT SUPPORT

[0001] This invention was made with government support under W81XWH2110469, W81XWH22C0059, and HT94252310392 awarded by The Department of Defense. The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] The present invention relates in general to determination of a brain or central nervous system (CNS) specific abnormal neurological condition of an individual such as a brain / CNS injury and in particular to measuring a quantity of brain specific biomarkers in sources, such as saliva, capillary blood (wet and dried blood, dried serum, or dried plasma samples) plasma or serum from venous blood collection or cerebrospinal fluid (CSF), to detect, monitor, diagnose, prognosticate, predict, differentiate, aid in the treatment of the abnormal condition, or a combination thereof. The ability to correlate samples from difference patient fluid sources, as well as correlating for patient age allows for diagnosis and tracking of healing of brain / CNS injury event or brain / CNS disorder initiation in field-, hospital-, and home-based environments.BACKGROUND OF THE INVENTION

[0003] Recent statistics from the Centers for Disease Control and Prevention (CDC) indicate that there are - 214,000 TBLrelated hospitalizations in the U.S. annually -representing 8% of all of the 2.8M new TBIs in the U.S. annually and 68% of the $80B hospitalization costs in the U.S. annually.1Survivors of TBI, particularly msTBI patients defined by the Glasgow Coma Scale (GCS) score of 3-12,2can suffer long-term or life-long health problems that may affect all aspects of their life=. Fall, firearm-related injuries, and motor vehicle accidents lead to most msTBIs, and ms TBI patients are the most likely to be readmitted within 30 days ofhospital discharge. This is particularly true for older patients due to falls and co-morbidities.3,5 14For example, 8.9% of 135,342 TBI patients hospitalized in 2014 were readmitted within 30 days of discharge: age, documentation of a fall, and intentional self-injury at the index admission were positively associated with readmission.3

[0004] The field of clinical neurology remains frustrated by the recognition that secondary injury to a central nervous system tissue associated with physiologic response to the initial insult could be lessened if only the initial insult could be rapidly diagnosed or in the case of a progressive disorder before stress on central nervous system tissues reached a preselected threshold. Traumatic, ischemic, and neurotoxic chemical insult, along with generic disorders, all present the prospect of brain damage. While the diagnosis of severe forms of each of these causes of brain damage is straightforward through clinical response testing and computed tomography (CT) and magnetic resonance imaging (MRI) testing, these diagnostics have their limitations in that spectroscopic imaging is both costly and time consuming while clinical response testing of incapacitated individuals is of limited value and often precludes a nuanced diagnosis. Additionally, owing to the limitations of existing diagnostics, situations under which a subject experiences a stress to their neurological condition such that the subject often is unaware that damage has occurred or seek treatment as the subtle symptoms often quickly resolve. The lack of treatment of these mild to moderate challenges to neurologic condition of a subject can have a cumulative effect or subsequently result in a severe brain damage event which in either case has a poor clinical prognosis.

[0005] There is also a growing appreciation that rapid intervention once a TBI is detected can greatly improve outcomes. The ability for a first responder to detect a TBI provides optimal clinical opportunities to limit the secondary inflammatory cascade that follows the injury. Increasing evidence suggests that TBI is also a risk factor for the development of age- associated neurodegenerative disorders including Alzheimer’s Disease (AD) and Parkinson'sDisease (PD). (Dams-O'Connor, K. et al.; Duan, Y. et al.; Lee, P.C. et al.; Sivanandam, T. M. et anon).

[0006] In order to overcome the limitations associated with spectroscopic and clinical response diagnosis of neurological condition, there is increasing attention on the use of biomarkers as internal indicators of change as to molecular or cellular level health condition of a subject. As detection of biomarkers uses a sample obtained from a subject and detects the biomarkers in that sample, typically cerebrospinal fluid, blood, or plasma, biomarker detection holds the prospect of inexpensive, rapid, and objective measurement of neurological condition. With the attainment of rapid and objective indicators of neurological condition allows one to determine severity of a non-normal brain condition on a scale with a degree of objectivity, predict outcome, guide therapy of the condition, as well as monitor subject responsiveness and recovery. Additionally, such information as obtained from numerous subjects allows one to gain a degree of insight into the mechanism of brain injury.

[0007] A number of biomarkers have been identified as being associated with severe traumatic brain injury as is often seen in vehicle collision and combat wounded subjects. Understanding how multiple biomarkers overlap and any correlations to injury severity remains unestablished. This lack of understanding is particularly prevalent with respect to traumatic injuries to the brain. Several of the current inventors pioneered the first “tandem’' blood biomarker test for TBI: neuronal ubiquitin C-terminal hydrolase-Ll (UCH-L1) and astrocytic glial fibrillary acidic protein (GFAP).15The FDA market-authorized the use of the biomarkers in lieu of computed tomography (CT) scans for mild TBI (concussion) within 12 hours of injury through the De Novo process in 2018.16This test fills an important gap in diagnosis of mild TBI by aiding in the identification of brain lesions and reducing the need for CT scans. Commercialization of the tandem test as an in vitro diagnostic (IVD) for mild TBI patients (Abbott Dx) on the point-of-care iSTAT immunoassay platform. FDA clearance of the iSTATplatform as a plasma-based test for use in lieu of CT scans for acute mild TBI (within 12 hours of injury), based on the ALERT-TBI study,17has also been obtained and is in clinical trials as an “aid-in-prognosis” for mild TBI. However, there are no FDA-approved blood tests for monitoring ms TBI in the post-acute phase from 1 day post-injury (dpi) and up to at least 6 mo post-injury. This gap in the monitoring of ms TBI is an urgent unmet need since most ms TBI patients require treatment and management (follow-up care) in hospital and post-hospital rehabilitative settings in the post-acute phase.

[0008] Analyses of a blast injury to a subject produced several inventive correlations between proteins and neuronal injury as an illustrative neurological condition. Neuronal injury is optionally the result of whole body blast, blast force to a particular portion of the body, or the result of other neuronal trauma or disease that produces detectable or differentiable levels of neuroactive biomarkers. Thus, identifying pathogenic pathways of primary blast brain injury (BBI) in reproducible experimental models is vital to the development of diagnostic algorithms for differentiating severe, moderate and mild (mTBI) from posttraumatic stress disorder (PTSD). Accordingly, a number of experimental animal models have been implemented to study mechanisms of blast wave impact and include rodents and larger animals such as sheep. However, because of the rather generic nature of blast generators used in the different studies, the data on brain injury mechanisms and putative biomarkers have been difficult to analyze and compare.

[0009] In spite of the extensive survey of brain specific proteins or autoantibodies thereto that become systemic in response to a brain specific abnormal neurological condition, usage of such markers has met with limited success in the field owing to a variety of issues such as sensitivity and the ability to obtain results in a clinically timely fashion.

[0010] The requirement for venous blood draws as a method of sample collection to detect biomarkers of abnormal neurological condition that are elevated in blood limits the usage ofsuch tests. As a result, care and / or treatment relevant data is either not available or delayed. As care and / or treatment of many abnormal neurological conditions has a limited timing window in order to avoid secondary injury, this has proven to be problematic.

[0011] Given the importance of GFAP as one of only two FDA-cleared biomarkers for TBI as well as being the most abundant and easy to measure biomarker for msTBI, the lack of information about the trajectory of other proteoforms of GFAP in post-acute msTBI is a significant knowledge gap.

[0012] Furthermore, there are no FDA-cleared capillary blood tests for TBI. This is surprising, given the ubiquity of dried (whole) blood spots for capillary blood sampling as part of prenatal testing and reports of longitudinal studies for non-CNS indications19, 20. However, there is sometimes a poor correlation between protein biomarker levels in dried blood spots due to irreversible adsorption and poor or irreproducible recovery.21Recently, some companies have met this challenge by sampling 3-4 drops of fingerprick capillary blood to reproducibly collect 10 pL of plasma onto a microfluidic dried plasma spot (DPS) sampling device in a manner similar to dried blood spot (DBS) sampling devices capable of measuring analytes with unprecedented volumetric precision from varying applied sample volumes and hematocrit levels.22In another advance, an internal quantitative DBS (iqDBS) sampling device, which delivers an exact volume of whole blood to a paper disc that is impregnated with a dried concentration of stable isotope internal standards, was created from DBS devices for improved quantification (sensitivity and accuracy) of derivatized amino acids using flow injection analysis with selected reaction monitoring (FIA-SRM) by a tandem mass spectrometer.23It has also been reasoned that immunoPCR-based quantification of protein biomarkers from fingerprick capillary blood from msTBI patients collected with DPS devices can be improved with the incorporation of a protein internal standard during manufacturing.

[0013] Thus, there exists a need for a process and an assay for providing improved measurement of brain specific abnormal neurological condition based on patient fluid samples other than venous blood. There also exists a need for a detection process and assay amenable to correlating these disparate fluid samples from a patient for an abnormal neurological condition with conventional venous or arterial blood drawn samples, as well as compensating for the age of the patient. There is additionally a need for building upon the collective evidence for surveilling and quantifying individual / cumulative effects of TBI with GFAP as a blood biomarker and building upon Applicant’ s discovery that GFAP is post-translationally modified by proteolytic processing by intracellular calcium-activated proteases such as calpain for truncation at the N-terminus to generate GFAP breakdown products termed herein as G* (or GBDP), which can, together with GFAP, serve as a novel pathological biomarkers of astrocyte debris clearance from brain tissue and CSF and promising biomarkers for msTBI.4, 18SUMMARY OF THE INVENTION

[0014] The present invention provides a process for determining an extent of a central nervous system (CNS) specific neurological condition in a subject. The process includes collecting a biological sample of a biofluid other than a venous or arterial blood draw from the subject into a collection device; measuring a quantity of first biofluid biomarker, a breakdown product (BDP) thereof, or a combination thereof from the sample; and correlating the quantity of first biofluid biomarker, BDP, or combination thereof with a quantity of first biofluid biomarker, BDP, or combination thereof from the venous or arterial blood draw from the subject to determine the extent of the CNS specific neurological condition in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 depicts a kit suitable for capillary blood draw according to the present invention;

[0016] FIG. 2 shows the three step process according to embodiments of the present invention;

[0017] FIG. 3 shows a graph of GFAP Spike Recovery in the described laboratory studies;

[0018] FIGS. 4A and 4B are graphs showing how Biomarker Levels in Capillary Blood from TBI Patients Correlate to Plasma Levels;

[0019] FIG. 5 is a graph showing how the ratio of GFAP to NfL in plasma and capillary blood corrects for a nonlinear recovery of biomarkers, and improves the R2 value, with n=25;

[0020] FIGS. 6A-6D are graphs showing that biomarker levels in Saliva from TBI Patients poorly correlate to Plasma levels;

[0021] FIGS. 7A-7D show top-ranked biomarker candidates revealed by immunoassaybased proteomics;

[0022] FIG. 8 shows how disease- and age-dependent effects of key biomarkers in CSF are Evident in TBI, MCI, and AD Patients;

[0023] FIG. 9 shows that statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with key biomarkers, including Serum NfL;

[0024] FIG. 10 shows that statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with Novel Endoplasmic Reticulum Biomarkers including Serum GB-2026;

[0025] FIG. 11 shows that statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI Patients is achievable with Novel Mitochondrial Biomarkers including Serum GB-0183;

[0026] FIG. 12 shows key biomarkers from dried plasma spots of EDTA whole blood (to simulate capillary blood) are stable at ambient temperature and humidity conditions;

[0027] FIG. 13 shows how biomarker levels in capillary blood from TBI patients are correlated to plasma levels: NfL;

[0028] FIG. 14 shows the statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI Patients is Achievable with Key Biomarkers of Serum NfL;

[0029] FIG. 15 shows the statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with novel mitochondrial biomarkers of Serum GB-0183;

[0030] FIG. 16 shows the statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with novel endoplasmic reticulum biomarkers of Serum GB-2026;

[0031] FIGS. 17A-17L shows graphs of Trajectory analysis of paired venous plasma and capillary blood samples in TBI patients;

[0032] FIGS. 18 A-l 8L show Correlation analysis of biomarker levels from TBI patients;

[0033] FIGS. 19A-19C show correlation analysis of biomarker levels from venous plasma and capillary blood collected on a DPS device from a subset of samples from Prospective Cohort #2;

[0034] FIGS. 20A-20D show trajectory and correlation analysis of ratios for GFAP / NfL for paired venous plasma and capillary blood samples in TBI patients;

[0035] FIGS. 21A-21L show trajectory and correlation analysis of ratios for GFAP / NfL, G* / NfL, and G* / GFAP for paired venous plasma and capillary blood samples in TBI patients;

[0036] FIGS. 22A-22L show venous plasma levels of GFAP / NfL, G* / NfL, and G* / GFAP for TBI patients; and

[0037] FIGS. 23A-23C show Baseline G* (Gstar) levels, unlike GFAP and NfL levels, do not increase with age in plasma samples from healthy controls (n = 60).DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention has utility in the diagnosis, care, and management of brain specific abnormal neurological conditions in general, and in particular, to traumatic brain injury (TBI) and (TBI-induced) Alzheimer’s disease (AD) and Alexander disease, in which a GFAP mutation is implicated in white matter deterioration. The subject invention also has utility in detecting brain specific abnormal neurological condition through detection of temporal blood biomarkers in capillary blood samples (wet and dried blood, plasma, and serum) from fingerprick blood collection, including proteins, metabolites, lipids, mRNAs, DNA, cells, microRNAs, and / or autoantibodies thereto. Abnormal neurological conditions can result from neurological trauma that illustratively results from percussive, blast or impact injuries or those resulting from ischemia, or disease. With resort to a microfluidics device or dried sample assays, a detection system is provided that is amenable to usage in the field, at home, and / or during patient transport as well as in the hospital. The present invention additionally has utility as a highly scalable and cost-effective sampling and testing solution for brain injury and health in pre-hospital, hospital, and post-hospital environments. The invention combines (i) point-of- care, minimally invasive sampling methods that use saliva or capillary blood with (ii) laboratory-based, high sensitivity immunoassays for determining the levels of acute, subacute, and chronic biomarkers from susceptible brain cell types.

[0039] The present invention provides early detection and monitoring of subclinical evidence of disease activity (sEDA), before, during, and / or after clinical evidence of disease activity (EDA) occurs.

[0040] The ability to use a blood sample drawn from capillaries, as compared to a venous or arterial source affords ease of collection advantages that are attractive for field-, hospital-, and home-based environments. The difficulties associated with capillary blood draw samplesinclude small sample volumes and correlation of detection results with venous or arterial draw blood samples. The usage of dried blood, dried plasma, and / or dried serum spots as a sample facilitates transport and storage, but adds complexity to testing as biomarker recovery, solubility, protection, and degradation must be considered along with the correlation of concentrations of any biomarker detected in capillary blood to concentrations of the biomarker in a venous or arterial (tube) blood.

[0041] FIG. 1 depicts a kit, shown generally at 10 for a capillary blood draw. The kit 10 includes a lancet 12 for penetrating the skin to obtain a capillary blood draw. While capillary blood draws are routinely performed on a subject finger, it is appreciated that heels, forearms, earlobes are illustrative of other conventional sites for obtaining a capillary blood draw. An expressed drop of blood has a typical volume of 20 to 50 microliters. The resulting drop of blood is transferred directly or with resort to an applicator (not shown) to a porous substrate 14 or a microfluidic device 16. The porous substrate 14 in some inventive embodiments has known blood retention volume such that a unit area of the porous substrate 16 correlates to a known volume of blood. Typically, a square millimeter of porous substrate 16 retains between 1 and 20 microliters of the capillary blood, with the thickness and wickability of the substrate toward blood being significant factors in blood retention volume of the porous substrate 16. After absorption, the liquid component of the blood drop evaporates leaving the biomarkers and corpuscular components from the blood drop retained in the porous substrate. In some inventive embodiments, a surface coating 17 is present on the porous substrate 14 with channels therein sized to permit blood plasma constituents to pass therethrough while cellular blood components are preferentially excluded from entering the porous substrate 14. A proteinaceous standard 15 is present in some inventive embodiments to assess the quality of the capillary blood draw.

[0042] Alternatively, or in concert with the porous substrate 14, a microfluidic device 16 is provided in the kit 10. The microfluidic device 16 has an inlet 18 for receipt of a drop of blood as denoted by the curved inlet arrow thereto. A buffer inlet 20 is also provided to dilute the blood and convey the components thereof, including the biomarkers of interest through a channel system, shown in simplified form at 20. One or more outlets 22 and 24 are provided for exhausting waste, fractions containing biomarkers, of the like. It is appreciated that determination of the amount of a given biomarker in a sample of capillary blood provides clinically useful information about the nature of an abnormal neurological condition, still other information useful in treatment may be present in the detection of isoforms, the degree of splicing, phosphorylation, other chemical and post-translational modifications, mutations, or a combination thereof for in a given biomarker. Tau protein is exemplary of a biomarker that includes secondary information such as phosphorylation at specific amino acid residues. Elution of biomarkers from the microfluidic device 16 affords the option to further evaluate biomarkers through techniques not incorporated to the microfluidic device. A similar kit is provided for saliva with a spit tube or swab with an associated collection tube in lieu of the lancet 12.

[0043] In some inventive embodiments, a device is provided in the kit 10 that is a lightweight, non-refrigerated, scalable, cost-effective, minimally invasive dried plasma spot (DPS) sampling device. Such a device, as shown for example with reference to 14 or 16 is designed specifically for self or caregiver-assisted finger-prick capillary blood collection at home or in the field (e.g., a sporting event or austere military environment) or hospital. The ability for individuals with no training or limited training to collect a capillary blood sample contemporaneously with a possible TBI, TBI induced Alzheimer’s disease (AD) or other event indicative of a negative change in neurological condition enhances the likelihood of detecting one or more biomarkers that have a peak concentration in blood within 4 hours of injury therebyproviding important clinical information as to the types of cells implicated in the event. A colorimetric line appearance or change indicative of an abnormal neurological condition is exemplary of an inventive device in general and testing for G* in particular.

[0044] The present invention provides sensitivity immunoassays with low sample volume requirements: a temporal blood biomarker panel for capillary blood samples for biomarkers with either comparatively high concentrations or for which sensitive detection techniques exist. Specifically, and as shown in the Appendix, GFAP and breakdown products (BDPS) thereof, are potentially attractive biomarkers to be collected from biofluids other than venous or arterial blood draws, yet the correlation of concentration from these other sources to venous blood draw obtained samples is variable, The present invention solves this problem by correction of GFAP and breakdown products (BDPS) thereof by creating a ratio with another biomarker from the same sample source or even another biofluid other than venous or arterial blood draws. Suitable reference biomarkers include NF-L, Tau, pTaul81, GFAP to its BDP(s), CypD (also known as PPIF), and combinations thereof, or a metabolic breakdown product of any of the aforementioned.

[0045] Still another problem with resort to creating a standardized test for a biomarker is that brain specific biomarkers tend to become more prevalent in the circulatory systems of the patient as a function of subject age. Without intending to be bound to a particular theory tight junction and metabolic aging of tissues is believed to result in the blood-brain barrier (BBB) becoming more porous as a function of age. This is addressed through a number of techniques according to the present invention. These include building a library of samples from normal subjects and correlating for age and optionally lifestyle or metabolic factors that can cause a deviation in baseline biomarkers relative to chronological age. Such factors include smoking, free radical concentrations, environmental exposures. Measurement of mitochondrial biomarker serum GB-0183 levels in the subject, measurement of a metabolic age by conventiontechniques, comparing to a demographically codes sample library or combinations thereof are all suitable for addressing this factor and therefore improving data quality for a brain biomarker in general, and GFAP and breakdown products (BDPS) thereof in particular.

[0046] In some inventive embodiments, inventive capillary blood draw is used in combination with conventional collection of venous or arterial blood samples for testing to establish the analytical comparability of an inventive sampling panel of temporal (acute, subacute, and chronic) CNS and non-CNS blood biomarkers with samples from both capillary blood collection (e.g., finger-prick capillary blood collection with dried plasma spot (DPS) sampling) and from venous blood collection. Some notable differences between the conventional venous or arterial blood samples and those of DPS include at least one of differential relative levels of recovery for a given biomarker, composite scores, and temporal profile thereof.

[0047] FIG. 2 shows the three step process according to embodiments of the present invention. First, venous or capillary blood is sampled, which allows prehospital, hospital, and post-hospital sampling for military and civilian TBI. Next, temporal blood biomarker panel testing is undertaken in a laboratory, which provides subphenotype and temporal information. Finally, an Evidence of Brain Health (EBH) or Molecular Evidence of Brain Injury (mEBI) score is provided, which Aids in the assessment of TBI and stage of injury.

[0048] According to the present invention, capillary blood is analyzed for a given biomarker from a dried blood spot or with a microfluidics device. Exemplary of a microfluidics device operative herein is that disclosed in US 20090053732 Al.

[0049] It is to be understood that in instances where a range of values are provided that the range is intended to encompass not only the end point values of the range but also intermediate values of the range as explicitly being included within the range and varying by the lastsignificant figure of the range. By way of example, a recited range of from 1 to 4 is intended to include 1-2, 1-3, 2-4, 3-4, and 1-4.

[0050] According to embodiments, a lightweight, non-refrigerated, scalable, cost-effective, minimally invasive Dried Plasma Spot (DPS) sampling device is provided that is designed specifically for medic-assisted blood collection in the field, as part of a single platform solution (SPS) to substantially improve the feasibility of sample collection for TBI patients prolonged field care (PFC), during transport from the field to a hospital, during their hospital stay, and during their time as out-patients away from a hospital setting. By maintaining a single DPS sampling platform throughout patient management and disposition, a composite score (threshold) is calculated without the need for difficult, to near impossible, bridging studies between point-of-care measurement solutions such as the iSTAT platform and more sensitive, hospital-based solutions such as the core lab Anility immunoassay platform. Moreover, unlike the iSTAT platform, DPS sampling eliminates the need for venipuncture collection or refrigerated transport and storage of blood specimens.

[0051] Embodiments of the present invention utilize DPS tests for biofluid TBI biomarkers. According to some inventive embodiments, 5-25 pL of plasma are placed onto a DPS from 3- 4 drops (70 pL) of finger prick capillary blood. According to embodiments, the present invention includes a dried plasma spot (DPS) sampling device. According to embodiments, the present invention uses a five-step sequence from serial sampling of 70 pL (3-4 drops) of finger-prick blood for capillary transport of preselected amount (e.g. 10 pL) of plasma to the DPS collection disk. The system collects 2 x 10 pl plasma from a finger stick of blood in the range of 35-55 % hematocrit. By exploiting the biomarkers and techniques of WO2016209147A1 and W02020050770A, all functionalities of the system including(l) blood pre-metering, (2) plasma extraction, (3) plasma metering, and (4) collection into a dried sample format are passively driven. The design allows for manipulating of liquids only throughcapillary forces enabling a completely autonomous multifunctional system. The system is constructed in some inventive embodiments using foil-based microfluidic technologies enabling high throughput manufacturing by roll-to-roll. This device is based on a successful dried blood spot sampling device that is capable of measuring analytes with unprecedented volumetric precision from varying applied sample volumes and hematocrit levels.

[0052] To demonstrate the feasibility of the present invention for early detection and monitoring of brain injury and health in different contexts of use, biomarker data was collected for saliva, capillary blood, plasma, serum, and CSF specimens prospectively collected from cross- and longitudinal-cohorts, from three observational clinical studies underway at five sites, as an interim readout. Additional biomarker data was collected from archived specimens from historical cohorts. Preliminary results show striking evidence for the potential utility of the solution - including minimally invasive high frequency longitudinal studies, detection of suspected TBI in ambulatory individuals, monitoring of organized sports and workplace, and diurnal sampling studies of glymphatic impairment and clearance. Capillary blood recoveries of 25-50%, compared to venous plasma levels, were observed at pg / mL ranges for numerous well-known biomarkers including p-taul81, tau, Api-42, NfL, GFAP, and IL6. The results show the discovery of several promising biomarkers in serum and CSF specimens. These results demonstrate that the present invention, combined with other modules and factors, such as cognitive assessments and age, help address the unmet medical needs of traumatic brain injury (TBI), mild cognitive impairment (MCI), and Alzheimer's disease (AD) patients in military and civilian populations.

[0053] FIG. 3 shows a graph of GFAP Spike Recovery in the described laboratory studies. To evaluate simulated capillary blood as a convenient alternative biofluid matrix, healthy EDTA whole blood and patient matched plasma was spiked with recombinant GFAP and recovery was measured comparing the sampling device to venous plasma with n = 4 for eachmatrix and dilution level. Recovery of Key Biomarkers from EDTA Whole Blood vs. Plasma is Reproducible but Nonlinear.

[0054] FIGS. 4A and 4B are graphs showing how Biomarker Levels in Capillary Blood from TBI Patients Correlate to Plasma Levels. Geriatric mild TBI from acute, subacute, and chronic geriatric military and civilian patients were sampled for venous plasma, capillary blood, and saliva. Matched capillary blood and plasma samples were tested on the same immunoassay panel for correlation with n = 26 for matched GFAP and n = 28 for matched NfL.

[0055] FIG. 5 is a graph showing how the ratio of GFAP to NfL in plasma and capillary blood corrects for a nonlinear recovery of biomarkers, and improves the R2value, with n=25.

[0056] FIGS. 6A-6D are graphs showing that biomarker levels in Saliva from TBI Patients poorly correlate to Plasma levels. Saliva does not correlate to matched plasma samples in GFAP, NfL, Tau, and pTaul81. Interestingly, Tau and pTau!81 levels are 2-4 orders of magnitude higher in saliva compared to plasma. Here n = 20-28.

[0057] FIGS. 7A-7D show top-ranked biomarker candidates revealed by immunoassaybased proteomics. Archived veteran CSF was grouped based on age, sex, and neurostatus.

[0058] FIG. 8 shows how disease- and age-dependent effects of key biomarkers in CSF are Evident in TBI, MCI, and AD Patients. GFAP, NfL, and a novel mitochondrial marker being explored by the applicant, are shown as a proof of concept of the increase in biomarker levels over time based on disease, injury, and time.

[0059] FIG. 9 shows that statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with key biomarkers, including Serum NfL. There are slight diverging trends between DAI- and DAI+ cohorts. No differences between DAI- and DAI+ at BL. Increased from BL to 24h and BL to 48h, with no change from 24h to 48h DAI- and DAI+. These results are adjusted for age, differences between cohorts seen at 24h and 48h. Significantclinical outcomes correlates determined with NPX values. Difference in poor outcome for DAI+ by time adjusted for age: / - 5.22, p = 0.0223.

[0060] FIG. 10 shows that statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with novel endoplasmic reticulum biomarkers including Serum GB-2026. These results show diverging trends between DAI- and DAI+ cohorts No differences are shown between DAI- and DAI+ at BL. Decreased from BL to 24h and BL to 48h, with no change from 24h to 48h for DAI; increased from BL to 24h, increased from BL to 48h for DAI+, with no change from 24h to 48h for DAI+. These results are adjusted for age, differences between cohorts seen at 24h and 48h. Significant clinical outcomes correlates determined with NPX values: Difference in DRS for DAI- by time adjusted for age: y2= 3.91, p = 0.0481.

[0061] FIG. 1 1 shows that statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI Patients is achievable with Novel Mitochondrial Biomarkers including Serum GB-0183. These results show no diverging trends between DAI- and DAI+ cohorts, no differences between DAI- and DAI+ at BL, no changes across all timepoints for DAI- and DAI+. These results are adjusted for age, differences between cohorts seen at 24h and 48h. The BL: p = 0.0705, 24h: p < 0.0001 and 48h: p < 0.0001. No significant clinical outcomes correlates determined with NPX values.

[0062] Analytical Figures of Merit from Dried Plasma Spots of EDTA Whole Blood (to Simulate Capillary Blood) are Compelling, being both accurate and precise. Analytical figures of Merit from Dried Plasma Spots of EDTA Whole Blood (to Simulate Capillary Blood) are Compelling for GFAP, showing dynamic range (4 or 5 PL) and linearity.

[0063] FIG. 12 shows key biomarkers from dried plasma spots of EDTA whole blood (to simulate capillary blood) are stable at ambient temperature and humidity conditions, GFAPAccelerated Stability Studies.

[0064] FIG. 13 shows how biomarker levels in capillary blood from TBI patients are correlated to plasma levels: NfL.

[0065] Recovery of key biomarkers from dried plasma spots of capillary blood (vs. Plasma) from TBI patients is reproducible: GFAP / NfL. Recovery of key biomarkers from dried plasma spots of capillary blood (vs. Plasma) from TBI patients (by Time Interval-Acute, Subacute, Chronic) is reproducible: GFAP / NfL.

[0066] The cohort included 1A = Deployed TBI Young, 3A = Deployed TBI Middle Age, 3D = MCI, 3E = MCI->AD, 3F = AD Early Onset, 3G = AD Old-Age.

[0067] FIG. 14 shows the statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI Patients is Achievable with Key Biomarkers of Serum NfL.

[0068] FIG. 15 shows the statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with novel mitochondrial biomarkers of Serum GB-0183.

[0069] FIG. 16 shows the statistically significant subphenotype stratification (DAI vs. non- DAI) of TBI patients is achievable with novel endoplasmic reticulum biomarkers of Serum GB-2026.

[0070] Additional correlations of GFAP and NfL blood biomarker results for prospectively collected paired venous plasma samples vs. DPS samples of capillary blood from longitudinal and cross-sectional cohorts of TBI patients are provided herein with numerous insights.

[0071] •Minimally invasive fingerprick collection of 3-4 drops of capillary blood with paper-based microfluidic devices for separation of plasma from red blood cells and storage in dried plasma spots (DPS) is a promising new tool for highly scalable and cost-effective capillary blood sampling and testing of biofluid biomarkers in TBI patients. Levels of GFAP and NfL from capillary blood samples from TBI patients were well-correlated to paired venous plasma samples at matched time points but highly variable (GFAP: Spearman’s p = 0.447- 0.647; NfL: p = 0.407-0.902). This is shown in FIGS. 17A-17H. FIGS. 17A-17L showtrajectory analysis of paired venous plasma and capillary blood samples in TBI patients.Venous plasma and capillary blood samples recovered from dried plasma spot s(DPS) are shown for glial fibrillary acidic protein (GFAP) and neurofilament light (NfL, synonymously denoted NFL and NF-L) levels with daily sampling for the first 48 hours and twice daily sampling thereafter until 14 days post injury (dpi). (A, B, E, F) Subjects A and B have mTBI (GCS = 13 and 14, respectively), and (C, D, G, H) Subjects C and D have msTBI subjects (GCS = 7 and 8, respectively). GFAP biomarker levels (A-D), and NfL biomarker levels (E- H), are displayed longitudinally. The GFAP / NfL ratio for each patient is shown for each patient (LL). (D,H) Timepoints boxed in red highlight clinical events: 1 - intracranial pressure (ICP) device insertion, 2 - seizure, 3 - ~12 hours post-seizure. Longitudinal monitoring of biomarker levels in paired samples is presented in absolute values (pg / mL) versus time from injury (hours). A trendline (loess, 95% confidence interval) is overlaid for each trajectory.

[0072] •Based on experience with technical replicates for individual patients (data not shown), failed capillary blood collection is the major source of variability that must be overcome to improve the correlation of biofluid biomarker results from capillary blood to venous plasma. However, the ratio of GFAP / NfL dramatically improved the correlation (FIGS. 17LL, FIG. 18, FIG. 19, FIG. 20). This is most simply explained by the normalization of poor vs. good capillary blood collections across biomarkers. Nonetheless, biological variability between biofluids has been previously observed for other capillary blood analytes. FIGS. 18A- 18L show correlation analysis of biomarker levels from TBI patients. Each point represents a paired venous plasma and capillary blood sample, recovered from a DPS device, for GFAP (top row) and NfL (bottom row) levels with daily sampling for the first 48 hours and twice daily sampling thereafter until 14dpi. (A, E) mTBI Subject A had a strong correlation between venous plasma and capillary blood biomarker levels Spearman rho correlation coefficients of C = 0.647 for GFAP and p = 0.902 for NfL. (B-D, F-G) Subjects B-D had moderate to strongcorrelations ranging from p = 0.407 - 0.677. Spearman rho correlation coefficients and p-values are shown with line of best fit and 95% confidence interval. P-values show significance (p <0.05) for all correlations, except (C). FIGS. 19A-19C show correlation analysis of biomarker levels from venous plasma and capillary blood collected on a DPS device from a subset of samples from Prospective Cohort #2. Each point represents a paired venous plasma and capillary blood sample, recovered from a DPS device, for GFAP and NfL levels from patients of various TBI severities, indicated in the legend. Pearson correlation coefficients represent a significant correlation in (A) GFAP (R2= 0.8424) and (B) NfL (R2= 0.8806). (C) The ratio of GFAP / NfL normalizes the data and improves the correlation coefficient in both Pearson (R2= 0.9076) and Spearman (r = 0.777) correlations. FIGS. 20A-20D show trajectory and correlation analysis of ratios for GFAP / NfL for paired venous plasma and capillary blood samples in TBI patients.

[0073] •G*, a novel GFAP proteoform of GFAP, is a useful biofluid biomarker in capillary blood and venous plasma samples from TBI patients due to its high relative abundance and distinct temporal profile (vs. GFAP and NfL) that mirrors pathological processes, as shown in FIG. 21. As above, ratios for GFAP / NfL, G* / NfL, and G* / GFAP provide evidence that ratios can be used for normalization of poor vs. good capillary blood collections across biomarkers, as shown in FIG. 22. This is important because it provides strong support for the novel strategy described herein. FIGS. 21A-21L show trajectory and correlation analysis of ratios for GFAP / NfL, G* / NfL, and G* / GFAP for paired venous plasma and capillary blood samples in TBI patients. FIGS. 22A-22L show venous plasma levels of GFAP / NfL, G* / NfL, and G* / GFAP for TBI patients.

[0074] •G* levels appear to be higher in capillary blood than venous plasma. Without intended to be bound to a particular theory, it is hypothesized that this finding is accounted for by one or more of: (i) true biodistribution differences between the biofluids, including levelsof G* and / or G* aggregation (see Insight #2 above); (ii) differences due to fingerprick vs. venous blood collection; and (iii) preanalytical differences arising from different levels of G* and / or G* aggregation following fingerprick versus venous collection. »G* is also less sensitive to age-related effects than GFAP or NfL (Fig. 10). This is true at both the low and high end of the age spectrum, where, unlike G*, plots of GFAP and NfL levels vs. age for healthy controls have a U-shape due to decreasing levels from birth up to age 4-5 from synaptic remodeling, followed by a nonlinear increase with age from adulthood to old age. G*, as a pathological biomarker, exhibits a relatively flat plot of levels across the age range in healthy controls. This has utility because thresholds for different age ranges might not need to be established for G*, unlike GFAP and NfL.

[0075] Based on the insights described above, embodiments of the present invention incorporate an internal protein standard during the manufacturing of DPS and DBS capillary collection devices to (i) unambiguously identify failed fingerprick capillary blood collections (failed collections) and (ii) improve the accuracy and precision of biomarker quantification.. Akin to the ratios described above, establishing the range of this internal standard allows for exploration of each proposed biofluid biomarker (e.g., GFAP, G*, NfL, IL-6) to internal standard ratio and to set QC thresholds for ratio(s) for eliminating failed collections. Since Applicant’s mAbs are harnessed to create new proprietary immuno PCR reagents for the Thermo PLA assays of GFAP and G*, ratios to these two biomarkers are the keys to successful commercialization of DPS and DBS capillary collection devices.

[0076] A proteinaceous standard operative herein illustratively includes a recombinant mutant of the biomarkers, lactate dehydrogenase, aspartate aminotransferase, a plasma soluble protein exogenous to the subject and having a molecular weight of from 4 to 100 kiloDaltons, green fluorescent protein, luciferase, amino acid tailed versions thereof (e.g. polyhistidine), andcombinations thereof. It is also appreciated that a proteinaceous standard is covalently bonded to a dye molecular in some inventive embodiments to facilitate spectroscopic studies.

[0077] According to embodiments, a new internal protein standard is incorporated during the manufacturing of the DPS or DBS device to (i) unambiguously identify failed fingerprick capillary blood collections (failed collections) and (ii) improve the accuracy and precision of biomarker quantification G*, GFAP, and other biomarkers.

[0078] According to embodiments, a diagnostic mAbs for glial fibrillary acidic protein (GFAP) and its N-terminally truncated proteolytic product G*, a novel pathological biomarker of astrocyte debris clearance from brain tissue and CSF.4 is harnessed to create new proprietary immunoPCR , and other types of immunoassays, reagents for GFAP and G*, and other biomarkers. Capillary blood collection devices, including DPS and DBS, are ideal for longitudinal studies because lightweight, fingerprick capillary blood sampling is decoupled from heavy sandwich ELISA, immunoPCR, or other immunoassay, instrumentation needed for hospital / central laboratory-based testing of low levels of brain and inflammatory biofluid biomarkers. In this way, the levels of biofluid biomarkers are inexpensively, safely, and frequently monitored in capillary blood and change over time in response to CNS injury / repair and care / recovery.

[0079] According to embodiments, capillary blood collection devices, including DPS and DBS, are combined with immunoPCR or other immunoassays for monitoring of confirmatory evidence of brain injury (EB1) or evidence of brain health (EBH) for msTBl according to FDA’s BEST biomarker definitions,24are further refined with patient age and days post-injury (dpi). This enhances the care of msTBl patients in several ways, including, but not limited to: Serving as the first highly sensitive and specific blood test for monitoring confirmatory EBI in msTBl patients in post-acute hospital (e.g., ICU) and out-of-hospital (e.g., rehabilitative care) settings and Informing on disposition (e.g., ICU / hospital discharge / readmission), rehabilitativecare, and return-to-work / play / activity decisions from 1 day post injury (dpi) to at least 6 months post-injury.

[0080] The present invention supports NIH’s Precision Medicine promise of delivering the right treatment, to the right patient, in the right timing,25and by the FDA’s 2023 guidance document entitled “Demonstrating Substantial Evidence of Effectiveness with One Adequate and Well-Controlled Clinical Investigation and Confirmatory Evidence Guidance for Industry”.

[0081] The present invention establishes the basis for conducting full clinical validation and seeking FDA clearance for a novel, highly scalable and cost-effective prototype DPS and DBS capillary collection devices to enable point-of-care sampling of biofluid biomarkers for postacute monitoring of confirmatory EBI in patients recovering from msTBI. Akin to the successes of high sensitivity troponin biomarkers for myocardial infarction26, 27and patientcentric sampling kits for COVID-19 testing,28sampling and testing of capillary blood biomarkers for TBI that improve data-driven prevention, identification, management, treatment, and care decisions for these patients. In the long term, this also supports clinical trials for novel interventions with disease modifying therapies towards further maximizing patient outcomes.

[0082] Patent documents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference to the same extent as if each individual document or publication was specifically and individually incorporated herein by reference.

[0083] While at least one exemplary embodiment has been presented in the foregoing description and attached appendix, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiment or exemplary embodiments are only examples, and are not intended to limit the scope, applicability, or configuration of thedescribed embodiments in any way. Rather, the foregoing description and incorporated references will provide those skilled in the art with a convenient roadmap for implementing the exemplary embodiment or exemplary embodiments. It should be understood that various changes may be made in the function and arrangement of elements without departing from the scope as set forth in the appended claims and the legal equivalents thereof.

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Claims

CLAIMS1. A process for determining an extent of a central nervous system (CNS) specific neurological condition in a subject comprising: collecting a biological sample of a biofluid other than from a venous or arterial blood draw from the subject into a collection device; measuring a quantity of first biofluid biomarker, a breakdown product (BDP) thereof, or a combination thereof from said sample; and correlating the quantity of first biofluid biomarker, BDP, or combination thereof with a quantity of first biofluid biomarker, BDP, or combination thereof from the venous or arterial blood draw from the subject to determine the extent of the CNS specific neurological condition in the subject.

2. The process of claim 1 wherein correlating is by measuring a second biomarker from the sample or another biofluid other than the venous or arterial blood draw.

3. The process of claim 2 wherein the second biomarker is NfL, a BDP if the GFAP is measured, or vice versa, or another BDP if the BDP is measured.

4. The process of claim 1 wherein the biological sample is a dried blood spot.

5. The process of claim 1 further comprising accounting for the age of the subject.

6. The process of claim 1 wherein the collection device includes a protein standard that is configured to identify failed collection of the biological sample.

7. The process of claim 1 wherein the first biofluid biomarker is at least one of GFAP, G*, NfL, IL-6.

8. The process of claim 7 wherein the first biofluid biomarker is G*, and not adjusting the correlation based on age of the subject.

9. The process of any one of claims 1 to 8 wherein the biological sample is a capillary blood sample.

10. The process of any one of claims 1 to 8wherein the collection device is a capillary blood collection device.

11. The process of any one of claims 1 to 8wherein the CNS specific neurological condition is a traumatic brain injury (TBI), TBLinduced) Alzheimer’s disease (AD) or Alexander disease.

12. The process of any one of claims 1 to 8 wherein the collection device includes a protein standard therein, the protein standard brought into contact with biological sample upon collection.

13. The process of any one of claims 1 to 8 wherein a first biomarker measured amount of the first biomarker is ratioed relative to second biomarker measured amount of the second biomarker to assess the validity of sampling of capillary blood as the biological sample.

14. A kit for performing the process of any one of claims 1 to 13 from capillary blood as the biological sample from a subject comprising: a lancet; anda porous substrate or a microfluidic device, the porous substrate or the microfluidic device inclusive of a proteinaceous standard operative to assess the correlation of the biological sample to venous blood from the subject.

15. The kit of claim 14 further comprising a colorimetric change indicative of an abnormal neurological condition in the subject.

Images