Enabling Quantification of Neuronal Biomarkers in Blood

An innovative platform, developed by Quanterix™, helps to determine the number of proteins present in various body fluids, including blood, at previously untraceable concentrations.

For medical researchers, this Single Molecule Array (Simoa™) technology will serve as an unparalleled tool for identifying low-abundance biomarkers and help expedite the development of a new class of diagnostic products that would be handy for detecting diseases at the early stage. The main differentiator of Quanterix™’s technology is unparalleled analytical sensitivity, which became feasible by its proprietary single-molecule detection technology.

The neuronal protein tau has been shown to play a key role in the diagnosis of Alzheimer’s disease, or AD. At present, cerebrospinal fluid (CSF) collection is required for measuring neuronal proteins, such as tau, since the concentrations of these molecules in peripheral circulation are less than the detection limit of traditional assays.

Therefore, the need for an invasive collection technique has reduced the application of CSF biomarkers. A method for determining the concentrations of neuronal proteins in the blood can considerably influence the diagnosis of various brain injuries, including neurodegenerative disease. Here, eliminating the necessity for costly, invasive, and time-intensive procedures is one such method. Until now, attempts to offer reliable and sensitive measurements in plasma and serum have not met with much success.1,2

Method

Quanterix™ developed the Simoa™ tau assay that utilizes a combination of antibodies that react with both phosphorylated and normal epitopes in the molecule’s mid-region. This makes the Simoa™ tau assay specific for all tau isoforms.

The assay’s limit of detection is 0.02 pg/mL, which is over 1000 times more sensitive when compared to traditional immunoassays (usually 30–60 pg/mL). Based on digital array technology, the assay utilizes BT2 monoclonal antibodies for detection (Pierce, now Thermo Fisher Scientific Inc., Waltham, MA, USA) and also uses the Tau5 monoclonal antibody for capture (Covance, Princeton, NJ, USA). A representative dose-response curve is shown in Figure 1.

Typical tau calibration curve. The Y-axis refers to the average number of enzymes per individual micro-bead captured in the microwells of the array. Each labeled immuno-complex corresponds to a single molecule of tau.

Figure 1. Typical tau calibration curve. The Y-axis refers to the average number of enzymes per individual micro-bead captured in the microwells of the array. Each labeled immuno-complex corresponds to a single molecule of tau.

Example 1, Brain Ischemia: Serum Tau Levels Predict Neurological Outcome After Hypoxic Brain Injury from Cardiac Arrest

Impartially determining the extent of brain injuries continues to be a major unmet clinical need. While clinical rating scales like Glasgow Coma Scale are valuable for grading the severity of an injury, and neuroimaging methods are handy for detecting the site and nature of the injury, they have reduced the potential to predict long- and short-term outcome. There are certain serum biomarkers that can speed up diagnosis in unconscious or sedated patients before neuroimaging. They can also stratify brain injuries for targeted intervention.

In the last 10 years, the potential usefulness of blood biomarkers for assessing brain injuries, including hypoxic brain injury, has been examined thoroughly. Due to the potential movement of increased CSF tau across the blood-brain barrier, it is believed that measurements of tau in blood would give an easy peripheral window into CSF/brain status.3

In a study, 25 men and women aged between 25 and 85 years (mean, 62 years) experiencing cardiac arrest were resuscitated. Following cardiac arrest, serial blood samples were obtained within six hours. As shown in Figure 2, time-dependent elevations of serum tau were noticed in all patients. Area-under-the-curve (AUC) helped in estimating tau appearance, which showed a statistically vital association with a six-month patient outcome (p < 0.01).

For the majority of patients who had poor outcomes, tau was observed in one or both of two main elevation peaks—the first one appearing soon after patient outcome and the second one occurring days later. In order to assess the importance of the delayed and initial tau peaks for patient outcome, AUCs were computed for the first 24 hours, the delayed tau peak, and the full-time course only.

Tau AUC and associated ROC curves for the secondary tau peak only. “Good” and “Poor” refer to the six-month outcome by Cerebral Performance Category assessment.

Figure 2. Tau AUC and associated ROC curves for the secondary tau peak only. “Good” and “Poor” refer to the six-month outcome by Cerebral Performance Category assessment.

The extent of the second peak seemed to hold slightly greater importance for long-term outcomes when compared to that of the first peak. In addition, serial measurements of the serum tau made by the Simoa™ tau assay were highly predictive of the neurological outcome after six months, anticipating good and poor outcomes with 100% specificity and 91% sensitivity, respectively.4

The study signified the initial high-sensitivity longitudinal examination of serum tau following acute hypoxic brain injury. The study was also the first to link serum tau with hypoxic brain damage evaluated by cerebral performance.

Example 2, Alzheimer’s Disease: Plasma Tau Levels are Increased in AD Patients

In clinical practice, AD diagnosis is made based on several factors. These include outcomes of neuropsychological and neurological tests, clinical features, and through the exclusion of other causes of dementia, such as frontotemporal and vascular dementia, or other neurological conditions, and diseases like Lewy body disease and Parkinson’s disease.

Owing to the clinical heterogeneity of AD, diagnosis of this medical condition continues to be vague until a post-mortem histopathological exam can be carried out. Moreover, the precision of such clinical diagnosis among skilled investigators at top medical centers is about 80% to 90%, and at the primary care level, this is even lower. This implies that better techniques are required to make a precise diagnosis. In this regard, guidelines have been recommended for what constitutes a valuable biochemical marker for AD.5

A well-defined biomarker that meets these guidelines would be expected to identify the underlying neuropathology and be verified in autopsy-validated AD cases. Biomarker sensitivity should be higher than 85% and specificity should exceed 80% to help differentiate AD from other forms of dementia. In addition, the ideal marker should be both reproducible and reliable across various laboratories, and the test should be inexpensive, noninvasive, and easy to perform.

Another major consideration in establishing the biomarker value is the ability to detect AD at the earliest stage of the disease before the appearance of cognitive symptoms. This medical condition is characterized histopathologically by the manifestation of extracellular deposits of amyloid beta-protein (Aß). These deposits form plaques and lead to intracellular accumulation of aggregated and hyperphosphorylated protein, which, in turn, forms neurofibrillary tangles.

Since the tau proteins and Aß have been linked with histopathologically validated disease, they have attracted a great deal of interest. Although several studies have been carried out to evaluate the levels of Aß42 and Aß40 in plasma or serum, they could not evaluate the tau levels so far. This is because existing assays do not have enough sensitivity to allow the detection of this biomarker. This problem could be resolved by developing an ultrasensitive test that would help in assessing the diagnostic value of tau in blood for the first time.

With the help of the Simoa™ tau assay, the relationship between AD and plasma tau levels was evaluated in a cross-sectional study of 25 cognitively normal controls, 75 patients with mild cognitive impairment (MCI), and 54 AD patients.6

Tau levels were observed to be considerably higher in AD patients when compared to both MCI and control patients, as shown in Figure 3. MCI patients who developed this condition at the time of follow-up had tau levels analogous to those of patients who had stable MCI and cognitively normal controls.

Elevated tau levels in plasma from patients with AD. A, Plasma levels of tau are elevated in patients with AD compared with cognitively normal controls and patients with MCI. B, MCI patients who developed AD (MCI-AD) during follow-up had baseline tau levels similar to those of patients with stable MCI (SMCI). C, There was no correlation between tau levels in plasma and CSF in any diagnostic group. Open circles, gray squares, and black triangles represent AD, MCI, and controls, respectively.

Figure 3. Elevated tau levels in plasma from patients with AD. A, Plasma levels of tau are elevated in patients with AD compared with cognitively normal controls and patients with MCI. B, MCI patients who developed AD (MCI-AD) during follow-up had baseline tau levels similar to those of patients with stable MCI (SMCI). C, There was no correlation between tau levels in plasma and CSF in any diagnostic group. Open circles, gray squares, and black triangles represent AD, MCI, and controls, respectively.

While the levels of plasma tau are increased in AD, the overlap occurring across diagnostic groups seems to reduce the utility of plasma tau as a stand-alone screening test. In addition, normal plasma tau levels in the MCI stage of AD signify that tau is a late biomarker, needing considerable injury before rising to unusual levels. The diagnostic groups did not show any association between the tau levels in CSF and plasma. This indicates that steady-state concentrations in both these body fluids are differentially controlled.

Example 3, Traumatic Brain Injury: Olympic Boxing is Associated with Elevated Plasma Tau Levels

Military personnel deployed in combat zones and athletes performing contact sports tend to suffer from traumatic brain injury, specifically mild traumatic brain injury (mTBI), which is a common occurrence. Even though evident symptoms of concussion/mTBI resolve quickly in most individuals, the overall burden of mTBI with respect to behavioral and cognitive impairments may be considerably underestimated.

An unmet diagnostic and prognostic need is recognizing patient populations at risk of developing the long-term consequences of TBI. Practical approaches to reliably and accurately differentiate between mTBI that is or is not linked with clinically important underlying brain damage are rather restricted.

At present, a head CT scan is considered the initial diagnostic test of choice but its inability to identify small subacute hemorrhages, axonal injury, white matter shearing, and minute contusions has limited its application. Although developments in MRI technology have enhanced the potential to identify mild brain injury, limited access, and high costs limit its clinical utility. There is an unmet clinical need for a means to determine circulating biomarkers that can identify mTBI, and recognize patients who need more assessment and treatment.

CSF biomarkers of brain injury—including proteins that point to neuroinflammation and glial, axonal, and neuronal damage—have already been established. Despite this fact, CSF cannot be readily accessed, specifically for patients suffering from mild and moderate TBI. Existing assays lack the required sensitivity to identify neuronal biomarkers present in peripheral fluids.

Boxers represent a population with an increased risk of TBI induced by mild, minimal, or severe/moderate damage. In addition, the cumulative effect of subconcussive rotational and translational punches to the head can lead to TBI. Such types of forces may lead to diffuse axonal injury and cortical damage. Moreover, chronic TBI may result from recurrent episodes of head injury, according to growing evidence.

In a study, 30 Olympic (amateur) boxers taking part in at least 47 bouts were evaluated against 25 controls.7 Blood was first obtained from the control group at one time and also from the boxer group within 1 to 6 days following a bout and following a rest period of at least 14 days. The Simoa™ digital assay was used to measure the Tau levels in plasma. It was seen that plasma tau was considerably elevated in the boxer group after a bout (2.46 pg/mL ± 5.1) when compared to the control group (0.79 pg/ mL ± 0.96) (see Figure 4).

Plasma tau is significantly elevated in boxers after a bout compared to controls. Peripheral blood was collected from the controls once. The boxers were tested 1–6 days after a bout (A) and after a rest period without exposure to bouts or training with blows to the head for at least 14 days (B).

Figure 4. Plasma tau is significantly elevated in boxers after a bout compared to controls. Peripheral blood was collected from the controls once. The boxers were tested 1–6 days after a bout (A) and after a rest period without exposure to bouts or training with blows to the head for at least 14 days (B).

However, plasma tau levels reduced dramatically in the boxer group after a resting period when compared to after a bout (p < 0.01). No major difference was observed between the boxer and control groups at follow-up. Outcomes suggest that a blood test can be used to diagnose boxing that may result in axonal injuries.

The study shows that Olympic boxing, as well as repetitive minimal head trauma, is linked with increased concentrations of plasma tau, even if there are no symptoms of minimal TBI or concussion. It also shows that the sensitivity of the Simoa™ tau assay serves as a novel tool for determining and examining the association between clinical presentation and neurological biomarkers.

Conclusion

The above data shows that the Simoa™ tau assay is the first platform to directly determine the appearance of tau in human plasma and serum. The consistent detection and measurement of tau levels in the blood could be clinically relevant for many significant applications. More studies using the Simoa™ tau assay may demonstrate the potential to determine other brain biomarkers readily in blood, offering a better understanding of diagnosis, observation, and treatment of various neurological conditions.

References

  1. Humpel C. Identifying and validating biomarkers for Alzheimer’s disease. Trends Biotechnol. 2011;29(1):26–32.
  2. Blennow K, Hampel H, Weiner M, Zetterberg H. Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nat Rev Neurol. 2010;6(3):131–44.
  3. Morris M, Maeda S, Vossel K, Mucke L. The many faces of tau. Neuron. 2011;70(3):410–26.
  4. Randall J, Mörtberg E, Provuncher GK, et al. Tau proteins in serum predict neurological outcome after hypoxic brain injury from cardiac arrest: results of a pilot study. Resuscitation. 2013;84(3):351–6.
  5. Mckhann GM, Knopman DS, Chertkow H, et al. The diagnosis of dementia due to Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 2011;7(3):263–9.
  6. Zetterberg H, Wilson D, Andreasson U, et al. Plasma tau levels in Alzheimer’s disease. Alzheimers Res Ther. 2013;5(2):9.
  7. Zetterberg H, Smith DH, Blennow K. Biomarkers of mild traumatic brain injury in cerebrospinal fluid and blood. Nat Rev Neurol. 2013;9(4):201–10.

About Quanterix

Quanterix is on a mission to change the way in which healthcare is provided by giving researchers the ability to closely examine the continuum from health to disease. In order to make this vision a reality, we brought together the most experienced management team, renowned scientists, industry leading investors and expert advisors, to form a collaborative ecosystem, united through the common goal of advancing the science of precision health.


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Last updated: Aug 13, 2019 at 3:54 AM

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