BLOOD READER SYSTEMS AND THERANOSTICS FOR BRAIN DAMAGE AND INJURY

Abstract

Blood and bodily fluid reader systems, including circulating biomarkers involving multiple mitochondrial releasates for providing real-time, at-the-scene objective indicia of individuals sustaining mild TBI.

Claims

1. A diagnostic reader system, comprising a portable reader unit configured to be carried by hand powered by a microprocessor and coupled to a portable computer configured to allow data transfer between said diagnostic reader and said portable computer, wherein said diagnostic reader contains a stylet or other unit to obtain a blood sample by finger stick or other bodily fluid sample and is operably connected to a disposable diagnostic device which has been contacted by the blood or other fluid sample taken from a person who may have suffered a blow or trauma involving the person's head, and wherein said disposable diagnostic device has: a. at least one first reactive surface which has a first type of biomolecular reagent bonded to it by which to measure a blood-borne concentration of at least one first mitochondrial releasate; b. at least one second reactive surface which has a second type of biomolecular reagent bonded to it by which to measure a blood-borne concentration of at least one second mitochondrial releasate; and, c. at least one third reactive surface which has a third type of biomolecular reagent bonded to it by which to measure a blood-borne concentration of at least one third mitochondrial releasate; and wherein said diagnostic reader system has data handling means selected from the group consisting of: i. displaying, in a manner visible to a user, both of the blood-borne concentrations of said first, second and third mitochondrial releasates; and, ii. transferring, to a portable computer, such as a smartphone or tablet, which has a display monitor, the blood-borne concentrations of said first, second and third mitochondrial releasates.

2. The diagnostic reader system of claim 1 wherein said first, second and third mitochondrial releasates are selected from the group consisting of: a. DNA segments (“native”) which are unaltered and specific to mitochondrial genes (mitochondrial DNA) and which do not normally occur in human nuclear DNA; b. fragments of mitochondrial DNA that have been degraded by oxidative radicals as a result of the brain injury in ways that normally are found, in humans, only in DNA fragments that have been released by mitochondria; c. proteins that are encoded by mitochondrial or nuclear DNA and concentrated in mitochondria prior to their rupture by the brain injury, including but not limited to; i. “high mobility group” (HMG) proteins; (a) High mobility group box 1 protein (HMGB1); (b) Transcription Factor for Mitochondria A (TFAM); ii. Cytochrome C oxidase; iii. Cyclophilin D; iv. Subunit 6 of ATP synthase; v. N-formyl peptides (N-FPs) and formyl peptide receptors (FPRs)

3. The diagnostic reader system of claim 1 wherein both of said first and second reactive surfaces are positioned in different locations on the single disposable diagnostic device, separated from the third reactive surface.

4. The diagnostic reader system of claim 1 wherein said diagnostic reader system is also configured to read data from different disposable diagnostic devices which have distinct areas that have been coated with biomolecular reagents that will indicate concentrations of one or more blood-borne human proteins selected from the group consisting of: a. apolipoprotein E; b. apolipoprotein A-1; c. one or more selected TAR DNA binding proteins; d. one or more cellular damage-associated molecular patterns (DAMPs) and damage-related proteins selected from the group consisting of; i. HMGB1 and TFAM (above as mitochondrial DAMPs) and; ii. angiotensin-converting enzyme serpin proteins, and plasminogen activator inhibitors; e. cytokines and/or other proinflammatory mediators; f. mRNA from genes which encode subunits of receptors that interact with cytokines or proinflammatory mediators and that include; i. thrombomodulin (THBD); ii. endothelial cell protein C receptor (ECPCR); iii. 5-hydroxytryptamine receptor 2A; iv. the serotonin transporter (SERT) or solute carrier family 6 (neurotransmitter transporter, 5-HTT); v. the human protein designated as SLC6A4; g. protein fragments normally found in receptors for thrombin and HMGB1 such as thrombomodulin (THBD); h. protein fragments normally found in receptors for advanced glycation end products (AGEs) such the receptor for AGEs (RAGE); and i. protein fragments normally found in pattern recognition receptors (PRRs) such as soluble Toll-like receptor (sTLR2 and sTLR4).

5. A system for monitoring or assessing mTBI in an animal or patient undergoing a therapeutic regimen for treatment of mTBI, the system comprising a portable reader unit configured to be carried by hand powered by a microprocessor and coupled to a portable computer configured to allow data transfer between said diagnostic reader and said portable computer, wherein said diagnostic reader contains a stylet or other unit to obtain a blood sample by finger stick or other bodily fluid sample and is operably connected to a disposable diagnostic device which has been contacted by the blood or other fluid sample taken from a person who may have suffered a blow or trauma involving the person's head, and wherein said disposable diagnostic device has: a. at least one first reactive surface which has a first type of biomolecular reagent bonded to it by which to measure a blood-borne concentration of at least one first mitochondrial releasate; b. at least one second reactive surface which has a second type of biomolecular reagent bonded to it by which to measure a blood-borne concentration of at least one second mitochondrial releasate; and, c. at least one third reactive surface which has a third type of biomolecular reagent bonded to it by which to measure a blood-borne concentration of at least one third mitochondrial releasate; and wherein said diagnostic reader system has data handling means selected from the group consisting of: i. displaying, in a manner visible to a user, both of the blood-borne concentrations of said first, second and third mitochondrial releasates; and, ii. transferring, to a portable computer, such as a smartphone or tablet, which has a display monitor, the blood-borne concentrations of said first, second and third mitochondrial releasates.

6. The system of claim 5 wherein said first, second and third mitochondrial releasates are selected from the group consisting of: a. DNA segments (“native”) which are unaltered and specific to mitochondrial genes (mitochondrial DNA) and which do not normally occur in human nuclear DNA; b. fragments of mitochondrial DNA that have been degraded by oxidative radicals as a result of the brain injury in ways that normally are found, in humans, only in DNA fragments that have been released by mitochondria; c. proteins that are encoded by mitochondrial or nuclear DNA and concentrated in mitochondria prior to their rupture by the brain injury, including but not limited to; i. “high mobility group” (HMG) proteins; (a) High mobility group box 1 protein (HMGB1); (b) Transcription Factor for Mitochondria A (TFAM); ii. Cytochrome C oxidase; iii. Cyclophilin D; iv. Subunit 6 of ATP synthase; v. N-formyl peptides (N-FPs) and formyl peptide receptors (FPRs)

7. The system of claim 5 wherein both of said first and second reactive surfaces are positioned in different locations on the single disposable diagnostic device, separated from the third reactive surface.

8. The system of claim 5 wherein said diagnostic reader system is also configured to read data from different disposable diagnostic devices which have distinct areas that have been coated with biomolecular reagents that will indicate concentrations of one or more blood-borne human proteins selected from the group consisting of: a. apolipoprotein E; b. apolipoprotein A-1; c. one or more selected TAR DNA binding proteins; d. one or more cellular damage-associated molecular patterns (DAMPs) and damage-related proteins selected from the group consisting of; i. HMGB1 and TFAM (above as mitochondrial DAMPs) and; ii. angiotensin-converting enzyme serpin proteins, and plasminogen activator inhibitors; e. cytokines and/or other proinflammatory mediators; f. mRNA from genes which encode subunits of receptors that interact with cytokines or proinflammatory mediators and that include; i. thrombomodulin (THBD); ii. endothelial cell protein C receptor (ECPCR); iii. 5-hydroxytryptamine receptor 2A; iv. the serotonin transporter (SERT) or solute carrier family 6 (neurotransmitter transporter, 5-HTT); v. the human protein designated as SLC6A4; g. protein fragments normally found in receptors for thrombin and HMGB1 such as thrombomodulin (THBD); h. protein fragments normally found in receptors for advanced glycation endproducts (AGEs) such the receptor for AGEs (RAGE); and i. protein fragments normally found in pattern recognition receptors (PRRs) such as soluble Toll-like receptor (sTLR2 and sTLR4).

9. The system of claim 5 wherein said diagnostic system comprises diagnosis by an original or modified version of a Glasgow Coma Score evaluation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 is a schematic of the reader/device system comprising the device (300) interacting via Bluetooth with the smartphone (200), interacting via the Internet with central server (100) and the biomarker panel modules (30, 32).

[0051] FIG. 2 is a graphic showing plasma levels of HMGB1 in normal male control and males suffering an acute myocardial infarction (AMI), to establish normal levels of <4 ng/mL.

[0052] FIG. 3a is a graphic showing plasma levels of HMGB1 in TBI patients at day 1, day 3, day 30 and day 90 after brain injury; FIG. 3b shows HMGB1 plasma levels is essentially an acute occurrence since 48% of all TBI patients had elevated levels at day 1; FIG. 3c shows that elevated HMGB1 levels are independent of plasma tau and GFAP levels in the same TBI patients, indicating they identify a separate endophenotype of TBI.

[0053] FIG. 4 is a graphic showing plasma levels of another “mitochondrial releasate”, transcription factor of mitochondria A (TFAM), an HMG protein similar to HMGB1, in mice subjected to mild contusion TBI (CCI) compared to “sham” controls (no CCI) but under anesthesia and surgical manipulation, after 72 hours.

[0054] FIG. 5 is a graphic of the same samples subjected to immunoprecipitation (IP) either to polyclonal rabbit antibody to TFAM or control (“no treatment”) antisera and subjected to immuno-blot.

[0055] FIG. 6 is a graphic of relative amounts of mtDNA in plasma of mice subjected to CCI and quantified by qRT-PCR indicating maximum amounts at 24 hours post-TBI but remaining elevated even one month post-TBI. Note that mtDNA circulates in control mouse plasma in these experiments.

DESCRIPTION OF THE INVENTION

[0056] As summarized above, this invention involves blood and fluid reader systems, materials, devices, and methods for evaluating the severity of traumatic head injuries, including concussions and other conditions, and thus, providing for removal of a player from the field, a trip to the hospital, a Medevac removal from the battlefield, or other steps to minimize brain damage. It further involves medicaments and methods of treatment of neuroinflammation involving chimeric compounds as disclosed herein. These systems, materials, devices, and methods are designed, in particular, for sites and situations where a relatively rapid determination must be made as to whether an athlete, soldier, accident victim, or anyone else who has been “stunned” or “shaken up”, by a blow to the head, a nearby explosion, or other comparable event, can rest for a brief period, “walk it off”, and then return to normal activity (such as to an ongoing athletic event, military patrol, etc.), or whether the person who suffered the blow to the head should receive prompt medical attention, to avoid or minimize lasting neurologic damage.

[0057] Accordingly, while the range and variety of uses for these types of diagnostic and evaluative materials, devices, and methods are not specifically limited to any particular class of events or activities, they should be regarded as being well-suited for use at events or in situations such as: [0058] a. athletic contests in which high-speed collisions between players occur, either as a normal part of the game (such as in boxing, extreme sports, football, hockey, etc.), or as less-frequent or incidental occurrences (such as in soccer, basketball, baseball, water polo, bicycle racing, etc.); [0059] b. during military activities, such as training exercises or patrols where accidents, ambushes, explosions, and similar events can occur; [0060] c. when “first responders” (which generally includes personnel who have been trained to respond to emergencies, such as police, firemen, ambulance attendants, etc.) arrive at the site of an accident or other incident or situation involving an apparent victim of a blow to the head or comparable trauma. [0061] d. These systems, methods and materials can utilize diagnostic bioreagents that are affixed to surfaces of computer-readable devices, which are designed to be analyzed by electronic machines referred to herein as readers. The types of devices used for these analyses are classified as either: [0062] i. discs, if they spin while being “read” (usually by a laser beam); or, [0063] ii. arrays, if they do not spin while being read. [0064] e. Such devices are available commercially, including from one or more of Affymetrix, Illumina, GE Healthcare, Applied Biosystems, Beckman Coulter, Eppendorf Biochip Systems, Agilent, Claros/OPKO Health, SynVivo/CFDRC and Qloudlab and their use is known to those of skill in the diagnostic art and can be targeted to analyze the quantity or concentration of a “mitochondrial releasate” as described herein. [0065] f. A “mitochondrial releasate” is firstly, a molecule (protein or nucleic acid) that is released from damaged or exploded mitochondria where it is, secondly, concentrated prior to its release. It might “originate” in the mitochondria or be transported there from the cell nucleus or cytoplasm, but is present and concentrated (about 5× or higher than that circulating in the blood) in the mitochondria prior to its release. [0066] g. The form and suffix of the word “releasate” is intended to be similar to other words that relate to or involve fluid behavior, including “condensates”, “leachates”, “absorbates”, etc. Accordingly, to qualify as a mitochondrial releasate, a mitochondrial molecule may, in certain circumstances, also be defined as one that is released by injured, damaged, and/or dying cells, into circulating blood, in quantities that enable those particular types of molecules to be used to analyze the extent and severity of cellular or tissue damage in response to a traumatic blow or similar injury. [0067] h. Not being bound hereby, there are at least the following types of candidate “mitochondrial releasates,” or mitochondrial DAMPs that are considered within the scope of the invention as offering substantial utility for evaluating the severity of a traumatic brain injury. These candidates can be summarized as follows:

[0068] 1. First Category: Mitochondrial DNA (mtDNA), as an Entirety

[0069] This category includes DNA sequences and segments that are specific to mitochondrial genes, as compared to nuclear genes (i.e., genes carried within the nucleus of a cell). The prefix “mt”, in “mtDNA”, specifically indicates that the DNA is of distinctly mitochondrial origin. Strands of mtDNA will bind, with affinity and specificity, to “complementary” DNA strands that have been affixed to the surface of a spinning disc or non-spinning array that is suited for processing and handling by the types of portable “reader” machines that are well-known and widely used for handling medical diagnostics and biochemical research. Accordingly, the types of methods and procedures that normally are used to perform “Southern blots”, which are standard and well-known types of tests that are done in biochemistry labs around the world, can be used to measure the levels of mtDNA in the blood of an athlete, soldier, accident victim, or other person of interest.

[0070] To optimize the utility of these types of tests, an athlete, soldier, or other person who is at elevated risk of a collision, attack, or other form of TBI preferably should be tested, at the beginning of a season, before being deployed to a combat zone, or at a comparable suitable time, to determine both: [0071] 1. a normal ratio between that person's mtDNA, and his/her nuclear DNA; and, [0072] 2. the quantity of mtDNA in that person's blood, under normal conditions.

[0073] If either (i) that ratio, or (ii) the concentrations of both mtDNA and nuclear DNA, in that person's blood, is found to be significantly altered, after a blow to the head or similar event, then the altered ratio and/or increased concentrations should be treated and regarded as a warning signal that the blow to the head may have created a concussion, and should be treated as a potentially serious neurologic problem that requires prompt medical attention.

[0074] Two specific mitochondrial genes for targeted analysis as described herein can be: [0075] a. the mitochondrial gene which encodes a protein called Cytochrome B oxidase, discussed below; and, [0076] b. the mitochondrial gene which encodes a protein called ATP synthase, subunit 6. [0077] c. 2. SECOND CATEGORY: DEGRADED MITOCHONDRIAL POLYNUCLEOTIDES [0078] d. In addition to testing for unaltered or native mtDNA, Southern blots can also be used to test for fragments of mtDNA that have been broken into pieces. That type of breakage is accelerated, in mtDNA as compared to nuclear DNA, by oxygen radicals (also referred to as oxygen free radicals, oxidative radicals, and “radical oxygen species” (ROS), which are found in abnormally high quantities inside the mitochondria (this is due to the fact that the mitochondria are the “cellular furnaces” where glucose (a sugar molecule) is “burned” (i.e., oxidized) to release its stored energy). Degraded mtDNA can also be assessed in a microfluidic chip using digital droplet-PCR (dd-PCR) [0079] e. 3. THIRD CATEGORY: CYTOCHROME C OXIDASE [0080] f. Cytochrome c oxidase is an enzyme which participates in the formation of “cytochrome c”, the so-called “death messenger” molecule, mentioned in the Background section. This protein enzyme is encoded by a “nuclear gene” (i.e., genes located on the chromosomes inside the nuclei of mammalian cells). However, once formed, these enzyme molecules migrate to (or are actively transported), i.e., they “translocate” to the mitochondria, and are taken inside the mitochondria to a point where almost no cytochrome c oxidase molecules remain as free molecules in the cytoplasm of a cell. Accordingly, cytochrome c oxidase provides an example of an enzyme (protein) that is found inside mitochondria, and which does not normally otherwise exist in substantial quantities in cytoplasmic fluid, or in blood samples in healthy people. [0081] g. Its presence and concentration can be detected by either: [0082] 1. assays or procedures that use monoclonal antibodies which bind specifically to cytochrome c oxidase; or, [0083] 2. assays which provide a substrate that is acted upon by Cytochrome C oxidase's enzymatic activity. [0084] h. 4. FOURTH CATEGORY: CYCLOPHILIN D (CypD) [0085] i. “Cyclophilin D” is another protein which is encoded by genes in the nuclei, but which actively migrates or is transported to mitochondria. Normally, it is affixed to mitochondrial membranes, as part of an organelle structure called the “mitochondrial permeability transition pore”. As such, it normally is located near the outer surfaces of the mitochondrial membranes, and it will be released, in a relatively rapid manner, if mitochondrial membranes in a cell are seriously damaged, to a point which causes them to rupture and break apart. It can be detected by assays that use monoclonal antibodies that bind specifically to cyclophilin D. [0086] j. 5. FIFTH CATEGORY: ATP SYNTHASE, SUBUNIT 6 [0087] k. This protein, which is encoded by mitochondrial genes, normally is found at significant quantities, in healthy tissue, only in mitochondria, rather than in cytoplasm or blood. It can be detected by assays that use monoclonal antibodies that bind specifically to subunit 6 of the ATP synthase complex. [0088] I. 6. SIXTH CATEGORY: HIGH MOBILITY GROUP BOX 1 PROTEIN (HMGB1) [0089] m. This protein, also encoded by a gene in a cell's nucleus, translocates to the mitochondria where it affects “mitochondrial quality control” or mitophagy. It is a prominent pro-inflammatory mediator; and, as amphoterin, it has been shown to be essential for normal brain development. As in other traumatic injuries, it is increased in brain and spinal cord tissue after injury, and is released into the bloodstream. [0090] n. 7. SEVENTH CATEGORY: TRANSCRIPTION FACTOR FOR MITOCHONDRIA A (TFAM) [0091] o. This protein transcription factor, also encoded by a nuclear gene, is responsible for mitochondrial biogenesis. TFAM is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. It is released from the brain after TBI. Like HMGB1, TFAM is a member of the high-mobility group (HMG) of proteins because it contains two HMG “boxes”. These factors make TFAM a promising “analyte” for use as described herein. [0092] p. OTHER BIOMOLECULES THAT CAN INDICATE INCREASED SUSCEPTIBILITY TO LONG-TERM PROBLEMS AFTER ONE OR MORE CONCUSSIONS [0093] q. In addition to the types of candidate molecules listed above, any of various additional types of bioreagents (such as monoclonal antibodies or strands of DNA which have specific binding affinity for any targeted biomolecules of interest in a person's blood) can be affixed to a diagnostic disc, array, or other device as described herein, to assist coaches, trainers, physicians, and others determine a certain person's susceptibility to neurodegenerative decay, and/or to mental or behavioral problems (such as lingering depression, disorientation, etc.), following a concussion or other head trauma. [0094] r. Accordingly, if a blood test is undertaken on a candidate athlete, either at the start of a season or at an important milestone in his or her career (such as when a candidate athlete is applying for an athletic scholarship to college, or is attempting to be selected for a professional sports team), it is considered within the scope of the invention to conduct a blood test and to use that same blood test to check for other, additional factors that may indicate a greater-than-normal susceptibility to long-term neurological, mental, or behavioral problems, if that candidate suffers a concussion. [0095] s. The following molecules are believed to be candidates for testing and analysis to determine higher-than-baseline levels of susceptibility to long-term neurological problems, following a concussion. [0096] t. Accordingly, the types of diagnostic discs and arrays disclosed herein, or otherwise known to those skilled in this art, can be designed to include reagents that will provide physicians, coaches, trainers, and other analysts with data that can indicate the blood-borne concentrations of any or all of the following biomolecules: [0097] i. Apolipoprotein E (APO-E); [0098] ii. TAR DNA binding protein (TDP-43; TARDBP); [0099] iii. Angiotensin-converting enzyme (ACE); other “serpin” proteins, such as protease nexin 1 (PN1; SERPINE1); neuroserpin (SERPINI1); and, plasminogen activator inhibitor 1 (PAI-1; Serpinel or SERPINE1); [0100] iv. cytokine genes and inflammatory mediators, and/or protein subunits from their receptors; examples include Interleukin-1 beta (abbreviated as IL-1β or IL-1 B), and tumor necrosis factor alpha (abbreviated as TNF-α, TNF-A, or simply TNF); [0101] v. Thrombomodulin (THBD), and endothelial cell protein C receptor (EPCR; PROCR); soluble forms of both (i.e, sTM and sEPCR); [0102] u. (6) 5-hydroxytryptamine (serotonin) receptor 2A (5-HT2A; HTR2A) and solute carrier family 6 (neurotransmitter transporter, 5-HT) and/or member 4 (SLC6A4; or SERT); [0103] v. (7) Certain types of “high mobility group” proteins, including “HMG box 1 protein” (HMGB1); and, [0104] w. (8) certain proteins referred to as “RAGE” proteins (which refers to “receptor for advanced glycation endproducts”, also given the acronym AGER), soluble form (i.e., sRAGE). [0105] x. As an example of the diagnostic blood reader system 100 of the invention, interfacing with a portable computer, smartphone or tablet 200, a portable handheld reader device 300 with individual protein or DNA detecting modules 30 and a stylet 40 for inducing a pinprick drop of blood (FIG. 1) is shown: In this depiction a reader 300 sold by Qloudlab, the Sceptre® device is used, but others are commercially available and known to those skilled in blood and other fluid analysis. This contains click-out modules 30 each with a separate sub-panel of the “mitochondrial releasates” 32 and click-out stylet 40 to prevent inter-patient contamination. [0106] y. In FIG. 2 levels of the DAMP (mtDAMP) HMGB1 in normal aged-matched controls (mean about 1.5) is compared with myocardial infarct patients (mean of almost 15). From the world literature normal blood values for this DAMP do not exceed 4 ng/mL.

[0107] Studies in Humans: [0108] a. HMGB1 is an example of a first “mitochondrial releasate”. It is a nucleus-encoded, non-histone nuclear protein that is translocated to mitochondria to effect mitochondrial quality control and mitophagy. In the example depicted, a highly sensitive and accurate HMGB1 ELISA Kit was used. Seventy-five days were analyzed (day; 44, day30 and 50 day90) banked repository plasma samples from mild-moderate TBI patients, for whom day 1 and then 3 subsequent post-injury samples were used. As shown in FIG. 2, HMGB1 levels are typically <4 ng/mL in control adult blood, whereas post-TBI (FIG. 3a), HMGB1 levels were markedly elevated (48%) within the first 24 hrs after TBI as shown in FIG. 3b. [0109] b. A number were >10 times normal plasma levels seen in FIG. 1. They subsequently declined but a few remained elevated at day 90. In most instances HMGB1 concentrations declined down to or close to baseline normal values by 30 days after injury, but in a number of patients a 2.sup.nd phase of elevated HMGB1 levels occurred at 90 days (FIG. 3a). The descriptive statistics for the plasma samples from post-TBI patients are shown in Table 1.

TABLE-US-00001 TABLE 1 Descriptive statistics for TBI samples ¤ N.sup.¤ Min.sup.¤ 1stQ.sup.¤ Median.sup.¤ Mean.sup.¤ 3rdQ.sup.¤ Max.sup.¤ ¤ HMGB1.sup.¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ d1.sup.¤ 75.sup.¤ 2.5.sup.¤  2.5.sup.¤  3.04.sup.¤  7.921.sup.¤  5.95.sup.¤ 71.7.sup.¤ ¤ d3.sup.¤ 31.sup.¤ 2.5.sup.¤  2.5.sup.¤  2.69.sup.¤  4.659.sup.¤   4.425.sup.¤ 21.1.sup.¤ ¤ d30.sup.¤ 44.sup.¤ 2.5.sup.¤  2.5.sup.¤  2.5.sup.¤  2.96.sup.¤ 2.5.sup.¤  11.07.sup.¤ ¤ d90.sup.¤ 50.sup.¤ 2.5.sup.¤  2.5.sup.¤  2.5.sup.¤   3.412.sup.¤ 2.5.sup.¤  26.44.sup.¤ ¤ GFAP.sup.¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ d1.sup.¤ 31.sup.¤ 0.sup.¤    3.795.sup.¤ 15.9.sup.¤   85.843.sup.¤ 83.9.sup.¤  863.3.sup.¤  ¤ d30.sup.¤ 32.sup.¤ 0.sup.¤     0.7415.sup.¤  1.325.sup.¤   1.8409.sup.¤  2.02.sup.¤  10.42.sup.¤ ¤ d90.sup.¤ 23.sup.¤ 0.282.sup.¤ 0.894.sup.¤ 1.35.sup.¤  1.919.sup.¤   2.195.sup.¤   6.43.sup.¤ ¤ Tau.sup.¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ d1.sup.¤ 32.sup.¤ 2.9.sup.¤  6.103.sup.¤ 9.56.sup.¤ 18.288.sup.¤ 16.3.sup.¤  85.5.sup.¤ ¤ d30.sup.¤ 32.sup.¤ 2.9.sup.¤  4.755.sup.¤  6.665.sup.¤ 10.678.sup.¤   9.047.sup.¤ 97.8.sup.¤ ¤ d90.sup.¤ 23.sup.¤ 2.18.sup.¤  3.94.sup.¤  5.72.sup.¤  6.007.sup.¤  6.97.sup.¤ 15.sup.¤   ¤ [0110] a. Also compared were the plasma concentrations of tau and GFAP proteins in the same study samples to the current HMGB1 values (Table 1). Concentrations of these proteins, tau for neuronal and GFAP astrocytic, respectively, required use of single-molecule technology (SIMOA, Quanterix) since they are in the pg/mL range. In contrast, HMGB1 bloodstream concentrations are 1000-fold greater, in the ng/mL range, and are read easily by a conventional bench-top or even point-of-care (POC) ELISA reader. These are graphically depicted in FIG. 3c. In addition to day1, day30 and day90, day3 was also analyzed for HMGB1 levels. Also measured were levels of HMGB1 in plasma from another cohort of TBI patients. Similar findings were found.