Elecsys Phospho-Tau (217P) Plasma

{0} [LOGO] FDA U.S. FOOD & DRUG ADMINISTRATION ### 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ### I Background Information: A 510(k) Number K261686 B Applicant Roche Diagnostics C Proprietary and Established Names Elecsys Phospho-Tau (217P) Plasma D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | SET | Class II | 21 CFR 866.5840 - Alzheimer's Disease Pathology Assessment Test | IM - Immunology | ### II Submission/Device Overview: A Purpose for Submission: New device B Measurand: Phospho-Tau (217P) (pTau217) C Type of Test: Fully automated, electrochemiluminescence immunoassay (ECLIA) ### III Intended Use/Indications for Use: A Intended Use(s): Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} See Indications for Use below. ### B Indication(s) for Use: The Elecsys Phospho-Tau (217P) Plasma assay is an in vitro immunoassay for the determination of the phospho-tau (217P) (pTau217) protein in human plasma from individuals aged 55 years and older. The assay is intended for use as an aid in identifying patients with amyloid pathology presenting with signs, symptoms, or complaints of cognitive decline associated with Alzheimer's disease. The Elecsys Phospho-Tau (217P) Plasma assay result must be used in the diagnostic pathway in conjunction with other clinical information. The electrochemiluminescence immunoassay “ECLIA” is intended for use on cobas e immunoassay analyzers. ### C Special Conditions for Use Statement(s): Rx - For Prescription Use Only ### D Special Instrument Requirements: cobas pro e 801 analyzer (K191899) ### IV Device/System Characteristics: ### A Device Description: Elecsys Phospho-Tau (217P) Plasma is an assay kit that includes a set of immunoassay reagents for the measurement of the phosphorylated Tau 217 protein in human plasma (K2-EDTA and K3-EDTA). The reagent components packaged in the cobas e pack are ready-for-use and are supplied in snap-cap plastic bottles compatible with the system. | Component | Volume | Contents | | --- | --- | --- | | M | Streptavidin-coated microparticles, 1 bottle, 7.2 mL | Streptavidin-coated microparticles 0.72 mg/mL; preservative | | R1 | Anti-pT217-Ab~biotin 1 bottle, 5.1 mL | Biotinylated monoclonal anti-pTau217 antibody (rabbit/mouse); buffer; preservative | | R2 | Anti-Tau-Ab~Ru(bpy)\( ^{^} \) 1 bottle, 5.1 mL | Monoclonal anti-tau antibody (mouse/human) labeled with ruthenium complex; buffer; preservative | | \( ^^Tris(2,2'-bipyridyl)ruthenium(II)-complex (Ru(bpy) \) | | | K261686 - Page 2 of 37 {2} The following materials are required for Elecsys Phospho-Tau (217P) Plasma, but sold and packaged separately: - CalSet Phospho-Tau (217P) Plasma (4 x 1.0 mL): pT217p Cal1: approx. 0.14 pg/mL (2 bottles, each for 1.0 mL) pT217p Cal2: approx. 2.00 pg/mL (2 bottles, each for 1.0 mL) - PreciControl Phospho-Tau (217P) Plasma (6 x 1.0 mL): PC pT217p1: approx. 0.17 pg/mL (2 bottles, each for 1.0 mL) PC pT217p2: approx. 0.70 pg/mL (2 bottles, each for 1.0 mL) PC pT217p3: approx. 1.12 pg/mL (2 bottles, each for 1.0 mL) ### B Principle of Operation: 1. Specimen collection and preparation Refer to the package insert of Elecsys Phospho-Tau (217P) Plasma. 2. Elecsys Phospho-Tau (217P) Plasma Elecsys Phospho-Tau (217P) Plasma is a serological, sandwich principal immunoassay to be used on the cobas e immunoassay family of analyzers with an 18-minute test time and include the following steps: a) First incubation: 60 μL of sample, a biotinylated monoclonal Tau antibody specific for phosphorylation at threonine 217 and a monoclonal Tau-specific antibody labeled with a ruthenium complex, Tris(2,2'-bipyridyl)ruthenium(II)-complex (Ru(bpy), react to form a sandwich complex. b) Second incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. c) The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell II M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. d) Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a leading calibration curve provided via the cobas link. The analyzer automatically calculates the analyte concentration of each sample in pg/mL. 3. Interpretation of Elecsys Phospho-Tau (217P) Plasma results Results of the Elecsys Phospho-Tau (217P) Plasma are reported by the instrument. The result must be interpreted by the laboratory professional according to the table below: K261686 - Page 3 of 37 {3} | Elecsys Phospho-Tau (217P) Plasma | Test Result | Interpretation | | --- | --- | --- | | pTau217 > 0.378 pg/mL* | Positive | A positive result indicates a high likelihood of amyloid pathology. This result, in the absence of clinical evaluation, does not establish a diagnosis of Alzheimer's disease. | | > 0.223 pg/mL pTau217 ≤ 0.378 pg/mL | Intermediate | An intermediate result is indeterminate for the presence of amyloid pathology. It is recommended to consider these patients for further testing. | | pTau217 ≤ 0.223 pg/mL | Negative | A negative result indicates a low likelihood of amyloid pathology. Further clinical investigation into other causes of cognitive decline is recommended for these patients. | | *Primary Care / Low-Prevalence Guidance: Prevalence may be low in primary care and a lower Positive Predictive Value (PPV) is expected in these low-prevalence settings. Therefore, results must be interpreted within the context of a comprehensive clinical assessment. Primary care patients with positive results near the upper cutoff may require further diagnostic evaluation or specialist follow-up to confirm or exclude the presence of amyloid pathology. | | | ### V Substantial Equivalence Information: #### A Predicate Device Name(s): Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio #### B Predicate 510(k) Number(s): K242706 #### C Comparison with Predicate(s): | Device & Predicate Device(s): | Device K261686 | Predicate K242706 | | --- | --- | --- | | Device Trade Name | Elecsys Phospho-Tau (217P) Plasma | Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio | | **General Device Characteristic Similarities** | | | | Intended Use/Indications For Use | Elecsys Phospho-Tau (217P) Plasma is an in vitro immunoassay for the determination of the phospho-tau (217P) (pTau217) protein in human plasma from individuals aged 55 years and older. | The Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio is an in vitro test using human plasma (K2EDTA) that combines the results of Lumipulse G pTau 217 Plasma and Lumipulse G β-Amyloid 1-42-N Plasma assays into a ratio of pTau 217 to β-Amyloid | K261686 - Page 4 of 37 {4} | Device & Predicate Device(s): | Device K261686 | Predicate K242706 | | --- | --- | --- | | Device Trade Name | Elecsys Phospho-Tau (217P) Plasma | Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio | | | The assay is intended for use as an aid in identifying patients with amyloid pathology presenting with signs, symptoms, or complaints of cognitive decline associated with Alzheimer's disease. The Elecsys Phospho-Tau (217P) Plasma assay result must be used in the diagnostic pathway in conjunction with other clinical information. The electrochemiluminescence immunoassay "ECLIA" is intended for use on cobas e immunoassay analyzers. | 1-42 concentrations using the LUMIPULSE G1200 System. The Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio is intended to aid healthcare providers to identify patients with amyloid pathology associated with Alzheimer's disease. The Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio is indicated for adult patients, aged 50 years and older, presenting at a specialized care setting with signs and symptoms of cognitive decline. A test result ≤ 0.00370 is a negative result which is consistent with patients who are unlikely to have amyloid pathology. These patients should be investigated for other causes of cognitive decline. A test result ≥ 0.00738 is a positive result which is consistent with patients who are likely to have amyloid pathology. This result does not establish a diagnosis of Alzheimer's disease or other cognitive disorders. A test result between 0.00371 and 0.00737 is an indeterminate result which is consistent with patients who are uncertain to have amyloid pathology. These patients should be considered for further testing. The Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio results must be interpreted in conjunction with other patient clinical information. This test is not intended as a screening or stand-alone diagnostic test. | | Assay Format | Two-step sandwich | Same | | Assay Cut-off | Two cut-offs | Two ratio cut-offs | | Assay Output | Negative, Intermediate, and Positive | Negative, Indeterminate, and Positive | | Total Duration of Assay | 18 minutes | 30 minutes | K261686 - Page 5 of 37 {5} | Device & Predicate Device(s): | Device K261686 | Predicate K242706 | | --- | --- | --- | | Device Trade Name | Elecsys Phospho-Tau (217P) Plasma | Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio | | **General Device Characteristic Differences** | | | | Analyte | Phosphorylated Tau217 (pTau217) | pTau217 and β-Amyloid (1-42) | | Assay Type | Electrochemiluminescence Immunoassay (ECLIA) | Chemiluminescent Enzyme Immunoassay (CLEIA) | | Assay Antibodies | Biotinylated monoclonal anti-pTau217antibody (rabbit/mouse) and monoclonal anti-Tau antibody (mouse/human) labeled with ruthenium complex; | pTau217: Anti-pTau217 monoclonal antibody (mouse)-coated particles and alkaline phosphatase (ALP)-labeled anti-Tau monoclonal antibodies (mouse) β-Amyloid 1-42: Anti-β-amyloid 1-42 monoclonal antibody (mouse)-coated particles and alkaline-phosphatase (ALP)-labeled anti-β-amyloid 1-42 monoclonal antibodies (mouse) conjugate | | Sample Volume | 60 μL | pTau217: 40 μL β-Amyloid 1-42: 40 μL | | Sample Type | Human K2-EDTA and K3-EDTA plasma and K2-EDTA plasma drawn in tubes containing separating gel | Human K2-EDTA plasma | | Instrument | cobas pro e 801 analyzer | LUMIPULSE G1200 System | | Traceability/Standardization | Internal standard for pTau217 | pTau217: Traceable to in-house reference calibrators β-Amyloid 1-42: Traceable to Lumipulse G β-Amyloid 1-42 CSF assay (DEN200027) | | Calibrators | CalSet Phospho-Tau (217P) Plasma: Cal1: approx. 1.0 pg/mL and Cal2: approx. 8.0 pg/mL | pTau217: Lumipulse G pTau 217 Plasma Calibrator Set: Cal1: 0 pg/mL, Cal2: 0.250 pg/mL, Cal3: 1.000 pg/mL, Cal4: 5.000 pg/mL and Cal5:10.000 pg/mL β-Amyloid 1-42: Lumipulse G β-Amyloid 1-42-N Plasma Calibrator Set: Cal1: 0 pg/mL, Cal2: 30 pg/mL, Cal3: 100 pg/mL and Cal4: 1000 pg/mL | | Measuring Range | 0.056–2.40 pg/mL | pTau217: 0.047 – 10.000 pg/mL β-Amyloid 1-42: 0.8 – 500.0 pg/mL | K261686 - Page 6 of 37 {6} | Device & Predicate Device(s): | Device K261686 | Predicate K242706 | | --- | --- | --- | | Device Trade Name | Elecsys Phospho-Tau (217P) Plasma | Lumipulse G pTau 217/β-Amyloid 1-42 Plasma Ratio | | No high-dose hook effect | Up to 24 pg/mL | pTau217: Up to 610 pg/mL β-Amyloid 1-42: Up to 2200 pg/mL | | Detection Limit | Limit of Blank = 0.047 pg/mL Limit of Detection = 0.056pg/mL Limit of Quantitation = 0.056 pg/mL | pTau217: Limit of Blank = 0.250 pg/mL Limit of Detection = 8.0 pg/mL Limit of Quantitation = 8.0 pg/mL β-Amyloid 1-42: Limit of Blank = 0.250 pg/mL Limit of Detection = 8.0 pg/mL Limit of Quantitation = 8.0 pg/mL | | Reagent Stability | Unopened: 12 months at 2–8°C On-board: 16 weeks | Unopened: 9 months at 2–8°C On-board: 30 days | | Sample Stability | 7 days at 2–8°C 2 days at 20–25°C 3 month at -20– -15°C Freeze only once | 3 hours on board 1 day at 2–8°C Not recommended to store at 23–28°C 1 month at -30– -10°C 1 month at -60°C Up to three freeze/thaw cycles | ### VI Standards/Guidance Documents Referenced: The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used: - CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition - CLSI EP06, 2nd ed.: Evaluation of the Linearity of Quantitative Measurement Procedures – Second Edition - CLSI EP07, 3rd ed.: Interference Testing in Clinical Chemistry; Approved Guideline – Third Edition - CLSI EP09c 3rd ed.: Measurement Procedure Comparison and Bias Estimation Using Patient Samples - CLSI EP12, 3rd ed.: Evaluation of Qualitative, Binary Output Examination Performance – Third Edition - CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition - CLSI EP25-Ed2A: Evaluation of Stability of In Vitro Medical Laboratory Reagents-Second Edition K261686 - Page 7 of 37 {7} - CLSI EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition - CLSI EP35, 1st ed.: Assessment of Equivalence or Suitability of Specimen Types for Medical Laboratory Measurement Procedures - First Edition - CLSI EP37, 1st ed.: Supplemental Tables for Interference Testing in Clinical Chemistry: Approved Guideline, First Edition ### VII Performance Characteristics (if/when applicable): #### A Analytical Performance: All results met the manufacturer's pre-determined acceptance criteria. ##### 1. Precision/Reproducibility: A study was conducted in accordance with the CLSI guideline EP05-A3 to evaluate precision including the reproducibility of the Elecsys Phospho-Tau (217P) Plasma using a panel of 16 human K2-EDTA plasma samples with pTau217 concentrations that span the analytical measuring range. The same 16-member plasma sample panel was used to evaluate (i) within laboratory precision, (ii) lot-to-lot precision, and (iii) site-to-site reproducibility (only 10 of the 16 plasma samples were included), as described below: ###### (i) Within-laboratory precision To evaluate within-laboratory precision, each panel member was tested in two replicates per run, two runs separated by two hours per day for 21 days at a single site, using one cobas pro e 801 analyzer and one reagent lot to yield a total of 84 replicates per sample. The samples were run in a randomized order on the analyzer. In addition, three control levels were run for run validity. The mean (pg/mL), standard deviation (SD) (pg/mL) and percent coefficient of variation (%CV) were calculated. The results are summarized in the table below. | | | | Within-Run | | Between-Run | | Between-Day | | Within-Laboratory | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | Plasma 1 | 84 | 0.145 | 0.006 | 3.9 | 0.002 | 1.7 | 0.002 | 1.7 | 0.007 | 4.5 | | Plasma 2 | 84 | 0.213 | 0.007 | 3.4 | 0.006 | 2.8 | 0.005 | 2.3 | 0.011 | 5.0 | | Plasma 3 | 84 | 0.215 | 0.007 | 3.1 | 0.005 | 2.5 | 0.006 | 2.8 | 0.010 | 4.9 | | Plasma 4 | 84 | 0.258 | 0.007 | 2.8 | 0 | 0 | 0.004 | 1.5 | 0.008 | 3.2 | | Plasma 5 | 84 | 0.351 | 0.007 | 1.9 | 0.004 | 1.1 | 0.004 | 1.2 | 0.009 | 2.5 | | Plasma 6 | 84 | 0.376 | 0.009 | 2.4 | 0.000 | 0 | 0.005 | 1.2 | 0.010 | 2.7 | | Plasma 7 | 84 | 0.459 | 0.007 | 1.6 | 0.006 | 1.4 | 0.005 | 1.2 | 0.010 | 2.2 | | Plasma 8 | 84 | 0.558 | 0.011 | 1.9 | 0 | 0.0 | 0.004 | 0.7 | 0.012 | 2.1 | | Plasma 9 | 84 | 0.581 | 0.010 | 1.7 | 0.005 | 0.9 | 0.003 | 0.5 | 0.012 | 2.0 | | Plasma 10 | 84 | 0.743 | 0.010 | 1.4 | 0.002 | 0.3 | 0.005 | 0.7 | 0.012 | 1.6 | | Plasma 11 | 84 | 1.07 | 0.016 | 1.5 | 0 | 0 | 0.005 | 0.5 | 0.017 | 1.6 | K261686 - Page 8 of 37 {8} | | | | Within-Run | | Between-Run | | Between-Day | | Within-Laboratory | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | Plasma 12 | 84 | 1.26 | 0.014 | 1.1 | 0.007 | 0.6 | 0.006 | 0.4 | 0.017 | 1.3 | | Plasma 13 | 84 | 1.76 | 0.016 | 0.9 | 0.00951 | 0.5 | 0.010 | 0.6 | 0.021 | 1.2 | | Plasma 14 | 84 | 1.98 | 0.020 | 1.0 | 0.008 | 0.4 | 0.009 | 0.4 | 0.023 | 1.2 | | Plasma 15 | 84 | 2.12 | 0.027 | 1.3 | 0.008 | 0.4 | 0.006 | 0.3 | 0.029 | 1.4 | | Plasma 16 | 84 | 2.31 | 0.026 | 1.1 | 0.007 | 0.3 | 0.014 | 0.6 | 0.030 | 1.3 | | PC pT217p 1 | 84 | 0.205 | 0.006 | 3.1 | 0.001 | 0.6 | 0.003 | 1.3 | 0.007 | 3.4 | | PC pT217p 2 | 84 | 0.687 | 0.010 | 1.4 | 0.007 | 1.0 | 0 | 0 | 0.012 | 1.7 | | PC pT217p 3 | 84 | 1.08 | 0.014 | 1.3 | 0 | 0 | 0.010 | 0.9 | 0.017 | 1.6 | | Panel members 1~4, 7, 15 and 16 were native samples, panel members 5, 6, 8- 14 were native samples spiked with pT217p calibrator peptide. PC=PreciControl | | | | | | | | | | | Qualitative agreement: A total of 84 replicates of each panel member were performed to evaluate qualitative agreement. The percentages of positive (>0.378 pg/mL) and negative (≤0.233 pg/mL) test results were calculated for each panel member. Results are summarized in the table below. | Panel member | Mean (pg/mL) | Total replicates | Qualitative agreement | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | Number of Positive Elecsys Phospho-Tau (217P) Plasma Results >0.378 pg/mL | % Positive Results | Number of Negative Elecsys Phospho-Tau (217P) Plasma Results ≤ 0.223 pg/mL | % Negative Results | | Plasma 1 | \( 0.145^A \) | 84 | 0 | 0 (0/84) | 84 | 100 (84/84) | | Plasma 2 | \( 0.213^A \) | 84 | 0 | 0 (0/84) | 68 | 81 (68/84) | | Plasma 3 | \( 0.215^A \) | 84 | 0 | 0 (0/84) | 68 | 81 (68/84) | | Plasma 4 | \( 0.258^A \) | 84 | 0 | 0 (0/84) | 0 | 0 (0/84) | | Plasma 5 | \( 0.351^B \) | 84 | 0 | 0 (0/84) | 0 | 0 (0/84) | | Plasma 6 | \( 0.376^B \) | 84 | 38 | 45.2 (38/84) | 0 | 0 (0/84) | | Plasma 7 | \( 0.459^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 8 | \( 0.558^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 9 | \( 0.581^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 10 | \( 0.743^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 11 | \( 1.07^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 12 | \( 1.26^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 13 | \( 1.76^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 14 | \( 1.98^C \) | 84 | 84 | 100 (84/84) | 0 | 0 (0/84) | | Plasma 15 | \( 0.145^A \) | 84 | 0 | 0 (0/84) | 84 | 100 (84/84) | | Plasma 16 | \( 0.213^A \) | 84 | 0 | 0 (0/84) | 68 | 81 (68/84) | | \( ^A \) Below cutoff;\( ^B \) Cut-off ± 20%;\( ^C \) Above cutoff | | | | | | | K261686 - Page 9 of 37 {9} #### (ii) Lot-to-lot precision To evaluate between-lot precision, each panel member was tested in five replicates per run, with a single run on each day for five days at a single site, using one cobas pro e 801 analyzer and three reagent lots, yielding a total of 75 replicates per sample for all lots combined. The samples were run in a randomized order on the analyzer. In addition, three control levels were included for run validity. The results are summarized in the table below. | | | | Within-Run | | Between-Day | | Between-Lot | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | Plasma 1 | 75 | 0.144 | 0.006 | 4.0 | 0.002 | 1.6 | 0.006 | 4.2 | 0.009 | 6.0 | | Plasma 2 | 75 | 0.203 | 0.006 | 3.1 | 0 | 0.2 | 0.012 | 6.1 | 0.014 | 6.8 | | Plasma 3 | 75 | 0.204 | 0.007 | 3.2 | 0.002 | 1.1 | 0.010 | 5.1 | 0.013 | 6.1 | | Plasma 4 | 75 | 0.255 | 0.007 | 2.6 | 0.001 | 0.2 | 0.010 | 3.7 | 0.012 | 4.6 | | Plasma 5 | 75 | 0.350 | 0.008 | 2.4 | 0.003 | 0.8 | 0.010 | 2.7 | 0.013 | 3.7 | | Plasma 6 | 75 | 0.364 | 0.007 | 2.1 | 0.004 | 1.1 | 0.017 | 4.5 | 0.019 | 5.1 | | Plasma 7 | 75 | 0.450 | 0.009 | 2.1 | 0 | 0.0 | 0.003 | 0.6 | 0.010 | 2.2 | | Plasma 8 | 75 | 0.566 | 0.008 | 1.5 | 0.006 | 1.1 | 0.015 | 2.7 | 0.019 | 3.3 | | Plasma 9 | 75 | 0.581 | 0.011 | 1.8 | 0.001 | 0.2 | 0.008 | 1.3 | 0.013 | 2.3 | | Plasma 10 | 75 | 0.738 | 0.011 | 1.5 | 0.002 | 0.2 | 0.009 | 1.2 | 0.015 | 2.0 | | Plasma 11 | 75 | 1.07 | 0.014 | 1.3 | 0.001 | 0.1 | 0.016 | 1.5 | 0.021 | 2.0 | | Plasma 12 | 75 | 1.24 | 0.015 | 1.2 | 0 | 0.0 | 0.024 | 1.9 | 0.028 | 2.3 | | Plasma 13 | 75 | 1.75 | 0.024 | 1.3 | 0.010 | 0.6 | 0.040 | 2.3 | 0.047 | 2.7 | | Plasma 14 | 75 | 1.95 | 0.023 | 1.2 | 0.003 | 0.2 | 0.040 | 2.0 | 0.046 | 2.4 | | Plasma 15 | 75 | 2.10 | 0.025 | 1.2 | 0 | 0.0 | 0.046 | 2.2 | 0.052 | 2.5 | | Plasma 16 | 75 | 2.29 | 0.029 | 1.3 | 0.002 | 0.1 | 0.052 | 2.3 | 0.059 | 2.6 | | PC pT217p 1 | 75 | 0.226 | 0.008 | 3.8 | 0.003 | 1.5 | 0.021 | 9.1 | 0.023 | 10.0 | | PC pT217p 2 | 75 | 0.705 | 0.011 | 1.5 | 0.005 | 0.7 | 0.0218 | 3.1 | 0.025 | 3.5 | | PC pT217p 3 | 75 | 1.09 | 0.015 | 1.4 | 0 | 0.0 | 0.0135 | 1.2 | 0.020 | 1.8 | | Panel members 1-4, 7, 15 and 16 were native samples, panel members 5, 6, 8- 14 were native samples spiked with pT217p calibrator peptide. PC=PreciControl | | | | | | | | | | | Qualitative agreement: A total of 75 replicates of each panel member were performed to evaluate qualitative agreement. The percentages of positive (>0.378 pg/mL) and negative ( \( \leq \) 0.233 pg/mL) test results were calculated for each panel member. Results are summarized in the table below. K261686 - Page 10 of 37 {10} | Panel member | Mean (pg/mL) | Total replicates | Qualitative agreement | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | Number of Positive Elecsys Phospho-Tau (217P) Plasma Results >0.378 pg/mL | % Positive Results | Number of Negative Elecsys Phospho-Tau (217P) Plasma Results ≤ 0.223 pg/mL | % Negative Results | | Plasma 1 | \( 0.144^A \) | 75 | 0 | 0 (0/75) | 75 | 100 (75/75) | | Plasma 2 | \( 0.203^A \) | 75 | 0 | 0 (0/75) | 73 | 97.3(73/75) | | Plasma 3 | \( 0.204^A \) | 75 | 0 | 0 (0/75) | 74 | 97.4 (74/75) | | Plasma 4 | \( 0.255^A \) | 75 | 0 | 0 (0/75) | 0 | 0 (0/75) | | Plasma 5 | \( 0.350^B \) | 75 | 0 | 0 (0/75) | 0 | 0 (0/75) | | Plasma 6 | \( 0.364^B \) | 75 | 11 | 14.7 (11/75) | 0 | 0 (0/75) | | Plasma 7 | \( 0.450^B \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 8 | \( 0.566^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 9 | \( 0.581^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 10 | \( 0.738^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 11 | \( 1.07^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 12 | \( 1.24^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 13 | \( 1.75^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 14 | \( 1.95^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 15 | \( 2.10^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | Plasma 16 | \( 2.29^C \) | 75 | 75 | 100 (75/75) | 0 | 0 (0/75) | | \( ^A \) Below cutoff;\( ^B \) Cut-off ± 20%;\( ^C \) Above cutoff | | | | | | | ### (iii) Site-to-site reproducibility: To evaluate site-to-site reproducibility, each panel member was tested in three replicates per run, two runs per day with a minimum of two hours idle time, for five days at three sites with one cobas pro e 801 analyzer at each site, using one reagent lot, yielding a total of 90 replicates per sample for all sites combined. The results are summarized in the table below. | | | | Within-Run | | Between-Run | | Between-Day | | Between-Site | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | Plasma 1 | 90 | 0.143 | 0.009 | 6.2 | 0 | 0.0 | 0 | 0.0 | 0.005 | 3.8 | 0.010 | 7.3 | | Plasma2 | 90 | 0.199 | 0.010 | 5.2 | 0 | 0.0 | 0.004 | 1.9 | 0.006 | 3.0 | 0.013 | 6.3 | | Plasma 3 | 90 | 0.202 | 0.008 | 3.8 | 0.002 | 1.0 | 0.004 | 1.9 | 0.004 | 1.8 | 0.010 | 4.8 | | Plasma 4 | 90 | 0.253 | 0.012 | 4.3 | 0.004 | 1.6 | 0 | 0.0 | 0.006 | 2.5 | 0.013 | 5.3 | | Plasma 5 | 90 | 0.343 | 0.010 | 2.9 | 0 | 0.1 | 0.003 | 0.8 | 0.009 | 2.5 | 0.014 | 3.9 | | Plasma 6 | 90 | 0.360 | 0.009 | 2.4 | 0.006 | 1.7 | 0 | 0.0 | 0.009 | 2.5 | 0.014 | 3.9 | | Plasma 7 | 90 | 0.452 | 0.010 | 2.3 | 0.005 | 1.2 | 0.006 | 1.4 | 0.003 | 0.6 | 0.013 | 3.0 | | Plasma 8 | 90 | 0.557 | 0.012 | 2.2 | 0.007 | 1.2 | 0.004 | 0.8 | 0.010 | 1.7 | 0.018 | 3.1 | | Plasma 9 | 90 | 1.26 | 0.024 | 1.9 | 0.017 | 1.4 | 0.018 | 1.4 | 0.023 | 1.9 | 0.042 | 3.3 | K261686 - Page 11 of 37 {11} | | | | Within-Run | | Between-Run | | Between-Day | | Between-Site | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | Plasma 10 | 90 | 2.31 | 0.035 | 1.5 | 0.016 | 0.7 | 0.023 | 1.0 | 0.056 | 2.4 | 0.072 | 3.1 | | PC pT217p 1 | 90 | 0.208 | 0.007 | 3.5 | 0.004 | 1.8 | 0 | 0.0 | 0.005 | 2.3 | 0.010 | 4.6 | | PC pT217p 2 | 90 | 0.694 | 0.010 | 1.5 | 0.008 | 1.1 | 0.011 | 1.5 | 0.012 | 1.7 | 0.020 | 2.9 | | PC pT217p 3 | 90 | 1.08 | 0.014 | 1.3 | 0.012 | 1.1 | 0.003 | 0.3 | 0.016 | 1.4 | 0.024 | 2.2 | | Panel members 1–5 were native samples and panel members 6-10 were native samples spiked with pT217p calibrator peptide. PC=PreciControl | | | | | | | | | | | | | Qualitative agreement: A total of 84 replicates of each panel member were performed to evaluate qualitative agreement. The percentages of positive (>0.378 pg/mL) and negative ( \( \leq \) 0.233 pg/mL) test results were calculated for each panel member. Results are summarized in the table below. | Panel member | Mean (pg/mL) | Total replicates | Qualitative agreement | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | Number of Positive Elecsys Phospho-Tau (217P) Plasma Results >0.378 pg/mL | % Positive Results | Number of Negative Elecsys Phospho-Tau (217P) Plasma Results ≤ 0.223 pg/mL | % Negative Results | | Plasma 1 | \( 0.143^A \) | 90 | 0 | 0 (0/90) | 90 | 100 (90/90) | | Plasma 2 | \( 0.199^A \) | 90 | 0 | 0 (0/90) | 87 | 96.7 (87/90) | | Plasma 3 | \( 0.202^A \) | 90 | 0 | 0 (0/90) | 90 | 100 (90/90) | | Plasma 4 | \( 0.253^A \) | 90 | 0 | 0 (0/90) | 2 | 2.2 (2/90) | | Plasma 5 | \( 0.343^B \) | 90 | 0 | 0 (0/90) | 0 | 0 (0/90) | | Plasma 6 | \( 0.360^B \) | 90 | 8 | 8.9 (8/90) | 0 | 0 (0/90) | | Plasma 7 | \( 0.452^B \) | 90 | 90 | 100 (90/90) | 0 | 0 (0/90) | | Plasma 8 | \( 0.557^C \) | 90 | 90 | 100 (90/90) | 0 | 0 (0/90) | | Plasma 9 | \( 1.26^C \) | 90 | 90 | 100 (90/90) | 0 | 0 (0/90) | | Plasma 10 | \( 2.31^C \) | 90 | 90 | 100 (90/90) | 0 | 0 (0/90) | | \( ^A \) Below cutoff\( ^B \) Cut-off ± 20%\( ^C \) Above cutoff | | | | | | | ### 2. Linearity: The linearity of the Elecsys Phospho-Tau(217P) Plasma on the cobas pro e 801 analyzer was evaluated in accordance with the CLSI guideline EP06-Ed2. Three high native K2-EDTA plasma samples spiked with CSF were each mixed in varying proportion with a low analyte plasma sample to create a series of nine dilutions of pTau217 concentrations that span across the measuring range of the assay. Each sample dilution was measured in one run with four replicates using one reagent lot and one cobas pro e 801 analyzer. For each sample, the mean value of the measured values, predicted value and the deviation from linearity were calculated. The linear range is defined by means of measurements for the lowest and highest sample levels and is confirmed if all sample levels are within the specifications for allowable deviation and precision. Percent deviations from linearity (%DL) were calculated K261686 - Page 12 of 37 {12} as differences between the observed values and the predicted values divided by the predicted values. A weighted regression analysis was performed to determine the predicted values. The table below summarizes the regression statistics for each sample separately and for the four pooled samples. The %DLs were within ±10% for each dilution level in all three samples. | Plasma Sample | Range (pg/mL) | Predicted Line | R2 | | --- | --- | --- | --- | | 1 | 0.0330 – 2.68 | Y = 1.054x + 0.0127 | 0.998 | | 2 | 0.0412 – 2.68 | Y = 1.010x + 0.0114 | 0.999 | | 3 | 0.0511 – 2.60 | Y = 1.012x + 0.0078 | 0.999 | | 1–3 | 0.0330 – 2.68 | Y = 1.024x | 0.999 | Linearity results support the measuring range claim of 0.056–2.40 pg/mL for the Elecsys Phospho-Tau(217P) Plasma. ### High-Dose Hook Effect The high-dose hook effect was evaluated for the Elecsys Phospho-Tau (217P) Plasma using three reagent lots on a cobas pro e 801 analyzer. To determine the hook concentration, two K2-EDTA plasma sample pools spiked with a high concentration stock of the calibrator pTau217 peptide were diluted with native plasma in 13 steps. Each sample level was measured in triplicates within one run. The measured counts were plotted against the expected sample concentrations. The data supports the claim that there is no high-dose hook effect up to 24 pg/mL. ### 3. Analytical Specificity/Interference: #### (i) Interference The effect of potential endogenous and exogenous interfering substances on the Elecsys Phospho-Tau (217P) Plasma assay was evaluated in accordance with the CLSI guidelines EP07, 3rd ed. and EP37, 1st ed. A panel of six human K2-EDTA plasma sample pools (two native and four spiked with the calibrator pTau217 peptide) with pTau217 concentrations spanning the measuring range of the assay including one sample near each of the two cutoffs were evaluated for interference with a panel of eight endogenous substances: bilirubin, biotin, hemoglobin, human serum albumin, IgG, IgM, IgA, and intralipid. A separate panel of six human K2-EDTA plasma (two native and four spiked) samples with pTau217 concentrations spanning the measuring range of the assay including the two cut-offs were tested with a panel of 43 exogenous substances: 17 common and 26 special pharmaceuticals listed in the table below. Native human plasma samples were used for the low-level samples with pTau217 concentrations ranging from 0.1 to 0.25 pg/mL. The high human plasma samples were spiked with the calibrator pTau217 peptide to achieve final pTau217 concentrations ranging from 0.233 pg/mL to 2.01 pg/mL. Each sample panel includes one plasma sample pool with pTau217 concentration around each assay cut-off. All plasma sample pools were divided into two parts: one was spiked with the potential interferent (i.e., the test sample) and the other (i.e., the control sample) without interferent was spiked with the respective amount of solvent used to create the interfering substances panel. The pTau217 levels in the test K261686 - Page 13 of 37 {13} and control samples were measured in five replicates with one lot of reagents in the same run on a cobas pro e 801 analyzer. The %interference was calculated by comparing measurements of the test and control samples. No significant interference was determined for %interference within ±10%. Non-significant interferences were observed for the Elecsys Phospho-Tau (217P) Plasma up to the concentrations of the potential interfering substances tested as shown in the tables below. | Endogenous Interferents | Interferent Concentration | | --- | --- | | Anti-Streptavidin antibodies^{5} | 100 μg/mL | | Bilirubin^{6} | 72.6 mg/dL | | Biotin^{7} | 1320 ng/mL | | HAMA (Fragment antigen-binding region-IgG) | 1200.0 μg/mL | | HARA (IgG) | 100 μg/mL | | Hemoglobin | 1100 mg/dL | | Human serum albumin | 7.7 g/dL | | Immunoglobulin G (IgG)* | 2.81 g/dL | | Immunoglobulin M (IgM) | 1.0 g/dL | | Immunoglobulin A (IgA)^{#} | 1.08 g/dL | | Intralipid | 2200.0 mg/dL | | Rheumatoid factor^{@} | 1320 IU/mL | | ^{5} Anti-Streptavidin antibodies were evaluated with five (one native and four spiked) K2-EDTAplasma with pTau217concentrations of 0.25, 0.4, 0.6, 0.8 and 2.0 pg/mL ^{&} Bilirubin consists of a mixture of unconjugated and conjugated forms ^{^} Biotin was tested at 3600 ng/mL. The data supports no interference up to 1320 ng/mL ^{*} IgG was tested at 7.7 g/dL. The data supports no interference up to 2.81 g/dL ^{#} IgA was tested at 1.6 g/dL. The data supports no interference up to 1.08 g/dL ^{$} HAMA (Human Anti-Mouse Antibodies) and HARA (Human Anti-Rabbit Antibodies) were assessed in 2 replicates in the same run using two (one native and one spiked) K2-EDTA plasma sample pools with pTau217 concentrations of 0.25 and 0.6 pg/mL ^{@} Rheumatoid factor was evaluated with five (one native and four spiked) K2-EDTA plasma sample pools | | | Exogenous Interferents | Interferent Concentration | | --- | --- | | Common drugs | | | Acetaminophen | 156.0 mg/L | | Acetylcysteine | 1660.0 mg/L | | Acetylsalicylic Acid | 435.0 mg/L | | Ampicillin-Na | 333 mg/L | | Ascorbic Acid | 25900 mg/L | | Cefoxitin | 750.0 mg/L | | Cyclosporine | 1.8 mg/L | | Doxycycline | 45.0 mg/L | | Heparin | 3300 IU/L | | Ibuprofen | 360.0 mg/L | | Itraconazole | 30.0 mg/L | | Levodopa | 7.5 mg/L | | Methyldopa | 22.5 mg/L | | Metronidazole | 123.0 mg/L | K261686 - Page 14 of 37 {14} | Exogenous Interferents | Interferent Concentration | | --- | --- | | Phenylbutazone | 321.0 mg/L | | Rifampicin | 75.0 mg/L | | Theophylline | 60.0 mg/L | | Special drugs | | | Adalimumab | 240.0 mg/L | | Albuterol | 0.045 mg/L | | Amlodipine | 0.08 mg/L | | Atorvastatin | 0.756 mg/L | | Clopidogrel | 45.0 mg/L | | Digoxin | 0.039 mg/L | | Donepezil | 30.0 mg/L | | Escitalopram | 0.192 mg/L | | Esomeprazole | 19.8 mg/L | | Fluticasone | 0.00126 mg/L | | Formoterol | 0.000273 mg/L | | Furosemide | 15.9 mg/L | | Galantamine | 250.0 mg/L | | Hydrochlorothiazide | 1.13 mg/L | | Insulin | 111.0 mU/L | | Lisinopril | 2.61 mg/L | | Losartan | 3.24 mg/L | | Memantine | 2.46 mg/L | | Metformin | 12.0 mg/L | | Metoprolol | 1.5 mg/L | | Montelukast | 4.45 mg/L | | Prednisone | 0.114 mg/L | | Rivastigmine | 45.0 mg/L | | Rivaroxaban | 2.7 mg/L | | Simvastatin | 1.68 mg/L | | Sitagliptin | 1.15 mg/L | #### (ii) Cross-reactivity The performance of the Elecsys Phospho-Tau (217P) Plasma assay in the presence of putative cross-reactants was assessed using one human K2-EDTA plasma sample pool with a low pTau217 concentration. The sample pool was divided into two aliquots: one was spiked with the respective putative cross-reactants at concentrations of 60.0 pg/mL and 66.0 pg/mL and the other aliquot without the cross-reactant served as the unspiked reference sample. The concentration of each cross-reactant was chosen to reflect maximum physiological concentration. Each sample was tested in five replicates on a cobas pro e 801 analyzer, and the mean value of each test sample was compared to that of the reference sample. The percentage of cross reactivity was calculated using the following formula: \[ \% \text {cross - reactivity} = [ (\text {mean of test sample - mean of reference sample}) / (\text {cross - reactant concentration}) ] \times 100 \] K261686 - Page 15 of 37 {15} The test concentration and mean %cross-reactivity value for each cross-reactant are summarized in the table below. | Cross-reactant | Cross-reactant concentration (pg/mL) | %Cross-reactivity | | --- | --- | --- | | Non-phosphorylated Tau | 60 | 0.00630 | | pTau181 | 60 | 0.0216 | | pTau205 | 60 | 0.0124 | | pTau212 | 60 | 0.0165 | | pTau231 | 60 | 0.00485 | The Elecsys Phospho-Tau (217P) Plasma showed no significant cross-reactivity (within ±1% difference of test from reference sample) up to 60 pg/mL for the pTau cross-reactants. To assess the specificity of the anti-pTau217 capture monoclonal antibody, a sequence alignment analysis was performed comparing the antibody's epitope region to peptide sequences surrounding all 35 potential phosphorylation sites on human Tau441. For each threonine site, an 11-amino acid window (five residues flanking on either side) was selected to enable robust epitope comparison. Sequence alignment showed that only the pTau217 peptide matched the capture antibody's epitope; all other phosphorylated threonine sites displayed insufficient sequence homology. The closest related epitopes identified were pTau205 and pTau181, which nonetheless exhibited low similarity to the target epitope. Cross-reactivity testing with pTau205 and pTau181 peptides along with pTau212 and pTau231 peptides revealed no cross-reactivity effects, confirming the high specificity of the anti-pTau217 capture antibody and validating that no cross-reactivity is expected across the entire Tau441 protein. # 4. Assay Reportable Range: The Elecsys Phospho-Tau (217P) Plasma assay has a measuring interval of 0.056–2.40 pg/mL. Values below the Limit of Quantitation (LoQ) are reported as <0.056 pg/mL. Values above the measuring range are reported as >2.40 pg/mL. # 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): # 1) Traceability Standardization for Elecsys Phospho-Tau (217P) Plasma was performed against an in-house reference material human Tau(207-227)[pThr217]amid. The purified reference material was absolutely quantified via amino acid analysis. The amino acid analysis was calibrated with the Certified Reference Material traceable to National Institute of Standards and Technology (NIST). # 2) Stability K261686 - Page 16 of 37 {16} a) Calibration (i) Lot Calibration Frequency A panel of 17 human K2-EDTA plasma samples with pTau217 levels covering the analytical measuring range was generated for lot calibration testing on the cobas pro e 801 analyzer. The low-level plasma samples were native (N=10 with pTau217 concentrations ranging from 0.15–0.55 pg/mL), and the middle (N=2 with pTau217 concentrations of 1.12 and 1.30 pg/mL) and high-level (N=5 with pTau217 concentrations ranging from 1.81–2.27 pg/mL) samples were spiked with the pTau217p calibrator peptide. Five of the 10 low-level samples are around (i.e., within ±20% of) the two assay cut-offs. A fresh Elecsys Phospho-Tau (217P) Plasma reagent lot was placed on the analyzer and calibrated on day 0. Baseline values for all samples were determined in duplicates on day 0 (time point 0). After 6, 12, and 14 weeks, the same panel of samples was tested in duplicates with a new reagent kit (stored at 2-8°C) of the same lot. Results of the samples on 6, 12 and 14 weeks were compared against the results of the samples on day 0. The specification (within ±10.0% deviation from time point 0) for lot calibration frequency was met for 14 weeks. The results support the lot calibration frequency claim of 12 weeks when using the same reagent kit kept at 2-8 °C. (ii) On-Board Calibration Frequency A panel of 17 pooled human K2-EDTA plasma samples with pTau217 levels covering the analytical measuring range was generated for on-board calibration stability testing on the cobas pro e 801 analyzer. The low-level plasma samples were native (N=10 with pTau217 concentrations ranging from 0.15–0.55 pg/mL), and the middle (N=2 with pTau217 concentrations of 1.12 and 1.30 pg/mL) and high-level (N=5; with pTau217 concentrations ranging from 1.81–2.27 pg/mL) samples were spiked with the pTau217p calibrator peptide. Five of the 10 low-level samples are around (i.e., within ±20% of) the two assay cut-offs. A fresh Elecsys Phospho-Tau (217P) Plasma reagent kit was placed on the analyzer and calibrated on day 0. Baseline values for the samples were determined in duplicates on day 0 (time point 0). The same samples were tested in duplicates after 8, 16, and 31 days with the same reagent kept in on-board condition using the calibration established on day 0. The mean recovery compared to the mean reference value (time point 0) was determined for all plasma samples using the calibration curve established on day 0. The specification (within ±10.0% deviation from time point 0) for on-board calibration stability was met for 31 days. The results support the on-board calibration frequency claim of 28 days when using the same reagent kit kept in on-board condition (10 ± 2 °C). b) Reagent (i) Shelf-life Stability K261686 - Page 17 of 37 {17} Reagent shelf-life stability of the Elecsys Phospho-Tau (217P) Plasma was determined on the cobas pro e 801 analyzer using three reagent lots and a panel of seven human K2-EDTA plasma samples with pTau217 concentrations covering the analytical measuring range of the assay. The five low-level plasma samples with pTau217 concentrations ranging from 0.110–0.460 pg/mL were native, and the middle and high-level samples with pTau217 concentrations of 1.24 and 2.02 pg/mL, respectively, were spiked with the pTau217p calibrator peptide. Three of the five low-level samples are around (i.e., within ±20% of) the two assay cut-offs. All samples were measured in one run in duplicates, and the mean value was calculated from each sample at time points 0, 10, 12, and 13 months for Lot#1 and at time points 0, 6.6 (for Lot #2)/ 9.0 (for Lot#3), 10, 12, and 13 months for Lots #2 and #3. At each timepoint, mean recovery of the test samples with respect to the initial measurement at timepoint 0 was evaluated. All samples tested with the three reagent lots were within specification (within ±10.0% deviation from timepoint 0). The results support that the Elecsys Phospho-Tau (217P) Plasma reagent kit can be stored unopened for up to 12 months when stored at 2–8°C. # (ii) On-Board Stability Reagent on-board stability for the Elecsys Phospho-Tau (217P) Plasma was tested on a cobas pro e 801 analyzer using one reagent lot and a panel of 15 human K2-EDTA plasma samples with pTau217 concentrations covering the analytical measuring range. The low-level samples (N=8 with pTau217 concentrations ranging from 0.15–0.55 pg/mL) were native, and the middle (N=2 with pTau217 concentrations of 1.12 and 1.30 pg/mL) and high concentration (N=5 with pTau217 concentrations ranging from 1.81–2.27 pg/mL) samples were spiked with the pTau217p calibrator peptide. Three of the eight low-level samples are around (i.e., within ±20% of) the two assay cut-offs. A freshly opened reagent kit was placed on the analyzer and calibrated. Baseline values for the sample panel were determined in duplicate at timepoint 0. After 4, 8, 12, 16 and 18 weeks on cobas pro analyzer (reagent kit kept at 10°C ± 2°C), frozen aliquots of the same samples were measured again in duplicate with the stressed kit. The mean recovery compared to the mean reference value (time point 0) was determined for all samples. The specification (within ±10.0% deviation from time point 0) for reagent on-board stability was met for 18 weeks. The results support that the Elecsys Phospho-Tau (217P) Plasma reagent kits can be stored on-board of the analyzer for up to 16 weeks. # (iii) Transport (Shipping) Stability Reagent transport stability for the Elecsys Phospho-Tau (217P) Plasma was tested on a cobas pro e 801 analyzer using three reagent lots and a panel of 17 human K2-EDTA plasma samples with pTau217 concentrations covering the analytical measuring range. The low-level samples (N=10 with pTau217 concentrations ranging from 0.15–0.78 pg/mL) were native, and the middle (N=2 with pTau217 concentrations of 1.12 and 1.30 pg/mL) and high concentration (N=5 with pTau217 concentrations ranging from 1.81–2.27 pg/mL) samples were spiked with the pTau217p calibrator peptide. The cobas e pack was stressed K261686 - Page 18 of 37 {18} in the original container for 7 and 8 days at temperature between 0°C and 25°C. The unstressed cobas e pack was kept at 2–8°C. Stability was assessed by testing the sample panel in duplicates and comparing mean recovery in the stressed and non-stressed reagent kits. The specification (within ±10.0% deviation from non-stressed reagent kit) for reagent transport stability was met for 1 week. The results support the reagent transport stability claim of 7 days at 25°C. # c) Specimen # (i) Specimen storage stability The stability of pTau217 in K2-EDTA was evaluated under various storage conditions on a cobas pro e 801 analyzer with a panel of 13 human plasma samples derived from whole blood that was freshly collected (never frozen) in accordance with the specimen collection and preparation procedure described in the package insert of the Elecsys Phospho-Tau (217P) Plasma. The following storage conditions were evaluated: room temperature (15°C–25°C), refrigerated (2°C–8°C) and frozen (-15°C–-20°C). The plasma samples in the panel were from patients representing the intended use population. The panel included four plasma samples in the low (≥ 0.0694 to ≤ 0.300 pg/mL) concentration range, six in the mid (> 0.300 to ≤ 0.600 pg/mL) concentration range, three in the high (> 0.600 to ≤ 2.4 pg/mL) concentration range, two around the lower cut-off (0.223 pg/mL), and five around the upper cutoff (0.378 pg/mL). All plasma samples were aliquoted. The baseline measurement (time point 0) was carried out using two aliquots for each plasma sample in the panel. Five replicates of each aliquot were tested, and the two aliquots were measured in separate runs. The baseline measurements were completed within six hours after the initial blood draw. Of the remaining aliquots, five aliquots were placed into each of the three storage conditions for the duration indicated in the table below. | Storage condition | Measurement Time Points (T)* | | | | | | --- | --- | --- | --- | --- | --- | | | T1 | T2 | T3 | T4 | T5 | | Room temperature (15°C–25°C) | 4 hours | 20 hours | 24 hours | 2 days | 3 days | | Refrigerated (2°C–4°C) | 1 day | 3 days | 6 days | 7 days | 8 days | | Frozen (-15°C– -20°C) | 3 weeks | 6 weeks | 9 weeks | 12 weeks | 13 weeks | | *Time since aliquot was placed in storage | | | | | | Linear regression analysis of the mean recoveries of aliquots at all time points under different temperatures was performed to determine the appropriate storage duration. All samples tested at each time point were within specification (within ±10% difference from baseline). The plasma sample stability claim for each storage condition is summarized in the table below. K2-EDTA and K3-EDTA Plasma Sample Stability K261686 - Page 19 of 37 {19} | Room temperature (15°C–25°C) | Refrigerated (2°C–4°C) | Frozen (-15 °C– -20°C) | | --- | --- | --- | | 2 days | 7 days | 12 weeks | # (ii) Specimen stability upon freezing and thawing One aliquot of each K2-EDTA plasma sample in the same panel used for specimen storage stability testing was used to evaluate the effect of repeated freeze-thaw (F/T) cycles on pTau217 measurement by the Elecsys Phospho-Tau (217P) Plasma. The aliquots were frozen at -80°C (-60°C to -90°C), after sample aliquoting for at least overnight and up to three days and thawed at room temperature (15°C–25°C) on a roller mixer for 15–30 minutes. Five replicates of each sample aliquot were tested on a cobas pro e 801 analyzer. The mean recoveries of all aliquots were within specification (within ±10% difference from baseline). The data show that pTau217 in K2-EDTA plasma samples is stable after three F/T cycles at -80°C. The package insert recommends freezing K2-EDTA plasma samples at -80°C only once. # 6. Detection Limit: The Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) studies were conducted in accordance with the CLSI guideline EP17-A2. The studies evaluated three lots of Elecsys Phospho-Tau (217P) Plasma on one cobas pro e 801 analyzer. A description of each study and the results obtained are summarized below: For the evaluation of LoB, six zero-level (blank) human K2-EDTA plasma samples, five from analyte-depleted and one native, were tested in six runs, distributed over five days, with two replicates per run for five samples tested with Lot #1 or ten replicates per run for one sample tested with Lots #2 and #3. A total of 60 replicates were obtained for all samples for each reagent lot. The LoB was determined as the 95th percentile of measurements obtained for each reagent lot. The LoB values from the three lots were 0.00878, 0.0227, and 0.0443 pg/mL. The claimed LoB is 0.047 pg/mL. For the evaluation of LoD, five low-level native K2-EDTA human plasma samples with target concentrations of pTau217 between 0.106 to 0.192 pg/mL were tested in six runs, distributed over five days, with two replicates per run to reach a total of 12 replicates per sample or 60 replicates for each reagent lot. The LoD values from the three lots were 0.0212, 0.0354, and 0.0544 pg/mL. The claimed LoD is 0.056 pg/mL. For the evaluation of LoQ, eight low-level K2-EDTA human plasma samples, five from partly analyte-depleted and three native, with pTau217 target concentrations between 0.037 pg/mL to 0.105 pg/mL were tested in five runs distributed over five days with five replicates per run to reach a total of 25 measurements per sample (200 measurements for all samples) for each reagent lot. The LoQ was estimated based on the lowest concentration of pTau217 which can be quantified with an intermediate precision of no more than 20% CV. The LoQ values from the three lots were 0.0333, 0.0392 and 0.0468 pg/mL. The claimed LoQ is 0.056 pg/mL. K261686 - Page 20 of 37 {20} # 7. Assay Cut-Off: The Elecsys Phospho-Tau (217P) Plasma assay cutoffs were determined by analyzing a total of 687 banked K2-EDTA plasma samples. The population was comprised of individuals from both primary care (N=194; 28.2%) and secondary/tertiary care settings (N=493; 71.8%). The mean age was 71.7 years (range: 61.0–86.0 years), consisting of 60.1% females and 38.9% males. Amyloid PET positivity was determined by a majority vote derived from the ratings of five or six individual expert readers. Of the 687 evaluable samples, 286 (41.6%) were PET positive and 401 (58.4%) were PET negative. The prevalence of PET positivity in the primary care and secondary/tertiary care settings is 21.1% (41/194) and 49.7% (245/493), respectively. The following Elecsys pTau217p cutoffs were selected: 0.378 pg/mL (upper Elecsys pTau217p cutoff) and 0.223 pg/mL (lower Elecsys pTau217p cutoff). # B Comparison Studies: # 1. Method Comparison with Predicate Device: Refer to Section C on 'Clinical Studies' below. # 2. Matrix Comparison: Sample matrix equivalence was evaluated in accordance with the CLSI guideline EP35, 1st ed. for samples collected with three different blood collection tube types: K2-EDTA, K3-EDTA, and K2-EDTA plasma gel separating tube (PST). A total of 59 sample pairs per tube type with pTau217 concentrations that span across the measuring range were tested in singleton with one reagent lot of Elecsys Phospho-Tau (217P) Plasma on a cobas pro e 801 analyzer. Samples with high pTau217 concentration were obtained by spiking of native plasma samples with the pT217p calibrator peptide. Passing-Bablok regression analysis was performed using pTau217 concentrations measured in samples collected with the primary tube (K2-EDTA without separating gel) compared to those in samples collected with each of the other tube types tested as summarized in the table below. | Candidate Tube | N of Total Samples | Range (pg/mL) | Slope | Intercept | Bias at cutoffs | | | --- | --- | --- | --- | --- | --- | --- | | | | | | | Absolute (pg/mL) | Relative (%) (95% CI) | | K3-EDTA vs K2-EDTA* | 59 | 0.0707–2.27 0.0677–2.28* | 1.023 | -0.0011 | cutoff=0.223 | | | | | | | | 0.00399 | 1.8% (0.2 – 3.3) | | | | | | | cutoff=0.378 | | | | | | | | 0.00751 | 2.0% (0.4 – 3.3) | | K2-EDTA plasma gel separating tube (PST) vs K2 EDTA* | 59 | 0.0721–2.28 0.0677–2.28* | 0.998 | 0.0031 | cutoff=0.223 | | | | | | | | 0.00274 | 1.2% (-0.2 – 2.1) | | | | | | | cutoff=0.378 | | | | | | | | 0.00247 | 0.7% | K261686 - Page 21 of 37 {21} | Candidate Tube | N of Total Samples | Range (pg/mL) | Slope | Intercept | Bias at cutoffs | | | --- | --- | --- | --- | --- | --- | --- | | | | | | | Absolute (pg/mL) | Relative (%) (95% CI) | | | | | | | | (-1.0 – 1.7) | | *The reference for all sample types was K2-EDTA plasma drawn into K2-EDTA plasma primary tubes | | | | | | | The package insert states that “Samples drawn in plasma separation tubes should be used immediately after sample processing and not be used after storage” ### C Clinical Evidence: #### 1. Clinical Sensitivity and Clinical Specificity: The clinical performance of the Elecsys Phospho-Tau (217P) Plasma assay was assessed in a retrospective study using amyloid PET visual read as the ground truth for brain amyloid status. This study utilized samples from two multicenter clinical cohorts: the Roche Diagnostic study (APPROACH cohort), and AACI study. The APPROACH cohort enrolled subjects from 16 clinical sites across the US and Europe, representative of primary and secondary/tertiary care settings. Enrolled individuals had cognitive complaints or objective memory impairment of unknown etiology. They were undergoing evaluations for Alzheimer’s disease and other causes of cognitive decline, for which the cause has been unknown, or would need referral for further cognitive evaluation. This cohort was representative of a broad, diagnostically naive patient population in early stages of cognitive impairment. Study AACI was a phase III clinical trial involving patients at a more diagnostically advanced stage of disease (mild stages of cognitive impairment or dementia). A sub-set of screening samples from this study, collected at US clinical sites representative of secondary/ tertiary care settings, were also included in the pivotal clinical performance study. The study enrolled 958 participants with valid Elecsys Phospho-Tau (217P) Plasma and amyloid PET results, with an average age of 71.1 years (range 55–85 years) who presented with cognitive complaints or impairment, subjective or objective, of unknown cause. A total of 876 (91.4%) subjects were from U.S. sites and 82 (8.6%) from non-US sites. A total of 234 (24.8%) subjects were from primary care sites and 720 (75.2%) from secondary and tertiary sites. The study subjects were categorized into four groups based on MMSE test results: MMSE 28-30 (N=295, 30.8%), MMSE 25-27 (N=363, 37.9%), MMSE 22-24 (N=227, 23.7%), and MMSE <22 (N=73, 7.62%). The study population consisted of 45.0% (431/958) males with a mean age of 71.0 years (range 55-85 years with a median age of 71 years), and 55% females (527/958) with a mean age of 71.2 years (range 55-85 years with a median 72 years). Regarding race, 84.7% were White, 11.5% were Black or African American, 1.6% were Asian, 2.2% identified as Other, and 0.10% were missing. Regarding ethnicity, 71.2% of participants were Not Hispanic or Latino, 19.6% were Hispanic or Latino, 0.522% were Not Reported, 0.104% were Unknown, and 8.56% of the participants' ethnicity was missing. The demographic and clinical characteristics of the patients across the four MMSE subgroups and according to PET scan results are presented in the table below. K261686 - Page 22 of 37 {22} | | MMSE | | | | Amyloid PET Visual | | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | | 28-30 | 25-27 | 22-24 | 20-21 | Positive | Negative | | | N(%Total) | 295(30.8%) | 363(37.9%) | 227(23.7%) | 73(7.62%) | 378(39.5%) | 580(60.5%) | 958(100%) | | Prevalence of Amyloid Positivity | 26.4%(78/295) | 38.8%(141/363) | 46.7%(106/227) | 72.6%(53/73) | | | 39.5%(378/958) | | Age [years] | | | | | | | | | Mean | 69.9 | 71.4 | 71.7 | 72.6 | 73.6 | 69.5 | 71.1 | | SD | 6.64 | 6.66 | 6.61 | 6.55 | 5.91 | 6.65 | 6.67 | | Median | 70.0 | 72.0 | 72.0 | 73.0 | 74.0 | 69.0 | 72.0 | | Age group | | | | | | | | | 55-70 yrs | n=149(50.5) | n=157(43.3) | n=98(43.2) | n=26(35.6) | n=108(28.6%) | n=322(55.5) | n=430(44.9%) | | 71-85 yrs | n=146(49.5) | n=206(56.7) | n=129(56.8) | n=47(64.4) | n=270(71.4%) | n=258(44.5) | n=528(55.1%) | | Sex | | | | | | | | | Male | n=136(46.1) | n=178(49.0) | n=90(39.6) | n=27(37.0) | n=174(46.0%) | n=257(44.3) | n=431(45.0%) | | Female | n=159(53.9) | n=185(51.0) | n=137(60.4) | n=46(63.0) | n=204(54.0%) | n=323(55.7) | n=527(55.0%) | | Race | | | | | | | | | White | n=264(89.5) | n=295(81.3) | n=183(80.6) | n=69(94.5) | n=363(96.0%) | n=448(77.2%) | n=811(84.7%) | | Black or African American | n=21(7.12) | n=48(13.2) | n=39(17.2) | n=2(2.74) | n=11(2.91%) | n=99(17.1) | n=110(11.5%) | | Asian | n=3(1.02) | n=10(2.75) | n=0(0%) | n=2(2.74) | n=2(0.529%) | n=13(2.24) | n=15(1.57%) | | Other | n=7(2.37) | n=9(2.48) | n=5(2.20) | n=0(0%) | n=1(0.265%) | n=20(3.45) | n=21(2.19%) | | Missing | n=0(0%) | n=1(0.27%) | n=0(0%) | n=0(0%) | n=1(0.265%) | n=0(0%) | n=1(0.104%) | | Ethnicity | | | | | | | | | Not Hispanic or Latino | n=241(81.7) | n=258(71.1) | n=132(58.1) | n=51(69.9) | n=301(79.6%) | n=381(65.7) | n=682(71.2%) | | Hispanic or Latino | n=20(6.78) | n=77(21.2) | n=78(34.4) | n=13(17.8) | n=36(9.52%) | n=152(26.2) | n=188(19.6%) | | Not Reported | n=1(0.33%) | n=2(0.55%) | n=1(0.44%) | n=1(1.37) | n=4(1.06%) | n=1(0.17%) | n=5(0.522%) | | Unknown | n=0(0%) | n=1(0.275%) | n=0(0%) | n=0(0%) | n=0(0%) | n=1(0.172%) | n=1(0.104%) | | Missing (n, %) | n=33(11.2) | n=25(6.89) | n=16(7.05) | n=8(11.0) | n=37(9.79%) | n=45(7.76) | n=82(8.56%) | | Mini-Mental State Examination | | | | | | | | | Mean | 28.7 | 26.1 | 23.1 | 20.7 | 24.9 | 26.3 | 25.8 | K261686 - Page 23 of 37 {23} | | MMSE | | | | Amyloid PET Visual | | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | | 28-30 | 25-27 | 22-24 | 20-21 | Positive | Negative | | | SD | 0.79 | 0.84 | 0.80 | 0.48 | 2.73 | 2.43 | 2.63 | | Median | 28.0 | 26.0 | 23.0 | 21.0 | 25.0 | 27.0 | 26.0 | | Q1–Q3 | 28.0–29.0 | 25.0–27.0 | 22.0–24.0 | 20.0–21.0 | 23.0–27.0 | 25.0–28.0 | 24.0–28.0 | | Min – Max | 28.0–30.0 | 25.0–27.0 | 22.0–24.0 | 20.0–21.0 | 20.0–30.0 | 20.0–30.0 | 20.0–30.0 | | Quick Dementia Rating System* | | | | | | | | | Mean | 3.57 | 4.29 | 4.80 | 5.25 | 4.12 | 4.09 | 4.10 | | SD | 2.24 | 2.42 | 2.94 | 2.33 | 2.41 | 2.52 | 2.49 | | Median | 3.00 | 4.00 | 4.00 | 5.25 | 4.00 | 3.50 | 3.50 | | Q1 – Q3 | 2.00 – 5.00 | 2.00 – 6.00 | 2.50– 6.50 | 3.38– 6.63 | 2.50 – 5.50 | 2.00 – 5.50 | 2.00 – 5.50 | | Min – Max | 0.50 – 12.0 | 0.50 – 11.5 | 0.50– 11.0 | 2.0 – 11.5 | 0.50 – 11.5 | 0.50 – 12.0 | 0.50 – 12.0 | | Missing (n, %) | n=69 (23.4%) | n=178 (49.0%) | n=136 (59.9%) | n=55 (75.3%) | n=227 (60.1%) | n=211 (36.4%) | n=438 (45.7%) | | Clinical Dementia Rating (CDR) Global * | | | | | | | | | 0 | n=68 (23.1%) | n=21 (5.79%) | n=3 (1.32%) | n=0 (0%) | n=20 (5.29%) | n=72 (12.4%) | n=92 (9.60%) | | 0.5 | n=149 (50.5%) | n=150 (41.3%) | n=69 (30.4%) | n=9 (12.3%) | n=105 (27.8%) | n=272 (46.9%) | n=377 (39.4%) | | 1 | n=5 (1.69%) | n=12 (3.31%) | n=16 (7.05%) | n=9 (12.3%) | n=24 (6.35%) | n=18 (3.10%) | n=42 (4.38%) | | 2 | n=0 (0%) | n=0 (0%) | n=0 (0%) | n=0 (0%) | n=0 (0%) | n=0 (0%) | n=0 (0%) | | 3 | n=0 (0%) | n=0 (0%) | n=1 (0.441%) | n=0 (0%) | n=0 (0%) | n=1 (0.172%) | n=1 (0.104%) | | Missing (n, %) | n=73 (24.7%) | n=180 (49.6%) | n=138 (60.8%) | n=55 (75.3%) | n=229 (60.6%) | n=217 (37.4%) | n=446 (46.6%) | | Clinical Dementia Rating–Sum of Boxes* | | | | | | | | | Mean | 1.31 | 2.06 | 2.87 | 4.94 | 2.50 | 1.75 | 1.97 | | SD | 1.30 | 1.40 | 1.69 | 1.83 | 1.89 | 1.46 | 1.63 | | Median | 1.00 | 2.00 | 2.75 | 4.50 | 2.00 | 1.50 | 1.50 | | Q1 – Q3 | 0 – 2.00 | 1.00 –3.00 | 1.50 – 4.00 | 3.75 –6.75 | 1.00 – 3.50 | 0.50 –2.50 | 1.00 – 3.00 | | Min – Max | 0 – 6.00 | 0 – 7.00 | 0 – 7.50 | 2.00– 8.00 | 0 – 7.00 | 0 – 8.00 | 0 – 8.00 | | Missing (n, %) | n=82 (27.8%) | n=188 (51.8%) | n=143 (63.0%) | n=56 (76.7%) | n=234 (61.9%) | n=235 (40.5%) | n=469 (49.0%) | | Education [years] | | | | | | | | | Mean | 15.8 | 15.3 | 13.9 | 15.4 | 15.4 | 15.0 | 15.2 | | SD | 3.47 | 5.66 | 4.92 | 10.2 | 5.76 | 5.26 | 5.46 | | Median | 16.0 | 15.0 | 14.0 | 14.0 | 16.0 | 14.0 | 15.0 | | Q1–Q3 | 13.0–18.0 | 12.0–17.0 | 12.0–16.0 | 12.0–16.0 | 12.0–18.0 | 12.0–17.0 | 12.0–17.0 | | Min–Max | 7.00–32.0 | 6.00–76.0 | 4.00–63.0 | 0–76.0 | 0–76.0 | 4.00–69.0 | 0–76.0 | | APOE4 status^ | | | | | | | | K261686 - Page 24 of 37 {24} | | MMSE | | | | Amyloid PET Visual | | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | | 28-30 | 25-27 | 22-24 | 20-21 | Positive | Negative | | | Carrier | n=105(35.6%) | n=138(38.0%) | n=86(37.9%) | n=41(56.2%) | n=253(66.9%) | n=117(20.2%) | n=370(38.6%) | | Non-carrier | n=184(62.4%) | n=218(60.1%) | n=140(61.7%) | n=31(42.5%) | n=119(31.5%) | n=454(78.3%) | n=573(59.8%) | | Missing (n, %) | n=6(2.03%) | n=7(1.93%) | n=1(0.441%) | n=1(1.37%) | n=6(1.59%) | n=9(1.55%) | n=15(1.57%) | | Clinical Cohort | | | | | | | | | APPROACH | n=226(43.5%) | n=185(35.6%) | n=91(17.5%) | n=18(3.5%) | n=151(29.0%) | n=369(71.0%) | n=520(100%) | | AACI | n=69(15.8%) | n=178(40.6%) | n=136(31.1%) | n=55(12.6%) | n=227(51.8%) | n=211(48.2%) | n=438(100%) | | Collection Site Location | | | | | | | | | U.S. | n=262(88.8%) | n=338(93.1%) | n=211(93.0%) | n=65(89.0%) | n=341(90.2%) | n=535(92.2%) | n=876(91.4%) | | Non-U.S.. | n=33(11.2%) | n=25(6.89%) | n=16(7.05%) | n=8(11.0%) | n=37(9.79%) | n=45(7.76%) | n=82(8.56%) | | Care Setting | | | | | | | | | Primary | n=79(26.8%) | n=101(27.8%) | n=55(24.2%) | n=3(4.11%) | n=51(13.5%) | n=187(32.2%) | n=238(24.8%) | | Secondary/Tertiary | n=216(73.2%) | n=262(72.2%) | n=172(75.8%) | n=70(95.9%) | n=327(86.5%) | n=393(67.8%) | n=720(75.2%) | | APOE4: Apolipoprotein E4; BMI: Body Mass Index; CSF: Cerebrospinal fluid; eGFR: estimated Glomerular Filtration Rate; Max: Maximum; Min: Minimum; MMSE: Mini-Mental State Examination; N / n: Number of subjects / Number of samples in the specific sub-category; PET: Positron emission tomography; Q1: 1st quartile; Q3: 3rd quartile; SD: Standard Deviation; U.S.: United States; yrs: years.* These cognitive assessments were systematically collected in the APPROACH cohort; they were not mandated by the screening protocol for the AACI cohort.^ Based on Elecsys Apolipoprotein E4 (protein) or genotyping. | | | | | | | | The four MMSE ranges were selected based on clinical staging frameworks. An MMSE score of 28–30 typically indicates objectively normal cognitive function on a gross screening instrument. Patients with early MCI can present with MMSE scores between 25 and 27. MMSE 22–24 captures the transition from mild cognitive impairment to mild dementia. MMSE scores <22 differentiate patients transitioning into more pronounced, moderate cognitive deficits from those in the mild/early symptomatic stages. The MMSE distribution for the APPROACH cohort was as follows: mean 26.6, median 27.0, range 20-30; while the MMSE distribution for the AACI cohort was: mean 24.7, median 25, range 20-28. To ensure the study population presented with subtle symptoms, the APPROACH study excluded subjects with a clinical diagnosis of moderate and severe dementia and/or MMSE score <20 and applied the following Quick Dementia Rating System (QDRS) inclusion criteria: - Subjects were required to have a QDRS score between 0.5 and 12 (inclusive). - Subjects were required to have a positive (impairment) score specifically in the Memory and Recall domain. K261686 - Page 25 of 37 {25} Except for active delirium or encephalopathy, participants with comorbidities frequently encountered in clinical practice were included unless the investigator deemed a condition likely to interfere with study procedures or long-term participation. ### Detection of amyloid pathology All subjects underwent amyloid PET scans using FDA approved amyloid tracers (18F-florbetapir, 18F-Florbetaben or 18F-Flutemetamol). The amyloid PET scans were randomly assigned, read and interpreted by six trained readers, three from two different vendors, each reading independent of each other's. Images that were rated positive by at least four readers had a positive majority vote and images that were rated positive by at most two readers had a negative majority vote (if at least one of the readers rates the image as non-readable, the PET scan result is rated as missing). In cases of a three-three split vote (three positive and three negative ratings), a tie-breaking method was established based on reader performance relative to CSF results: the reader with the lowest agreement with CSF results (the greatest outlier) was excluded, and the final assessment was determined by the majority vote of the remaining five readers. Final majority voting resulted in 378 (39.5%) positive and 580 (60.5%) negative amyloid PET reads. The independent readers were blinded to any clinical information, including the patient's clinical status, diagnosis, and plasma and/or CSF biomarker measurements. PET reads were conducted according to the approved instructions for use of the amyloid tracers. Across the full cohort, 20.7% (n = 198) of images received 6 positive reads, and 50.7% (n=486) received 0 positive reads; 6.26% (n=60) received 5 positive reads, and 6.47% (n=62) received 1 positive read; 3.86% (n=37) received 4 positive read and 3.24% (n=31) received 2 positive reads; 8.77% (n=84) of images received a 3-3 split read. The positive percent agreement (PPA) between readers was 83.6% on average (range: 44.3%-100%), the negative percent agreement (NPA) was 91.3% on average (range: 68.1%-100%), and the overall percentage agreement (OPA) was 87.3% on average (range: 74.4%-96.6%). The time difference between blood collection and PET imaging exhibited a mean of 37.7 days (SD=28.9) and a median of 30 days. Timing was consistent between PET positive (mean=32.0 days and median=27 days) and PET negative (mean=41.5 days and median=34 days) participants. ### Plasma sampling and analysis The analysis of plasma pTau217 among 958 participants with valid amyloid PET results revealed that amyloid PET Positive individuals had higher mean pTau217 levels (N=378; mean=0.633 pg/mL and median=0.582 pg/mL)) compared to PET Negative individuals (N=580; mean=0.202 pg/mL and median=0.172 pg/mL). ### Stability of banked clinical specimens A specimen stability study was conducted to demonstrate the integrity of clinical specimens stored frozen at -80°C for an extended duration. Results of the long-term stability study demonstrated that clinical specimens stored at -80°C were stable for up to 5.4 years. All archived K2-EDTA plasma evaluated in the pivotal clinical performance study were stored frozen at -80°C for four years after collection. ### Results: 1) Clinical Performance K261686 - Page 26 of 37 {26} To estimate clinical performance measures of Elecsys Phospho-Tau (217P) Plasma, test result was compared to the adjudicated amyloid PET scan for each patient. The adjudicated amyloid PET scan positive is called “Amyloid positive”. The data are summarized in the table below: | | Amyloid PET Visual | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Elecsys Phospho-Tau (217P) Plasma | Positive (> 0.378 pg/mL) | 285 | 37 | 322 | | | Intermediate (0.223< and ≤ 0.378 pg/mL) | 72 | 119 | 191 | | | Negative (≤0.223 pg/mL) | 21 | 424 | 445 | | | Total | 378 | 580 | 958 | The performance estimates of Elecsys Phospho-Tau (217P) Plasma are described by amyloid PET scan predictive value (indicated as Predictive Value, PV, percent of subjects with amyloid positive results) and frequency of positive test results from the tested samples (indicated as Frequency of Results, FR) for positive, intermediate, and negative test results. The agreement rates with visual read amyloid PET classification and predictive values for Elecsys Phospho-Tau (217P) Plasma at the pre-specified cut-offs of 0.225 pg/mL and 0.378 pg/mL and likelihood ratios are summarized in the table below. The prevalence of amyloid positivity based on amyloid PET was 13.1% in the study population. | | Amyloid PET Visual | | Predictive Value% (n/N) (95% CI)* | Frequency of Results % (n/N) | Likelihood Ratio (95% CI)** | | | --- | --- | --- | --- | --- | --- | --- | | | | Positive | | | | Total | | Elecsys Phospho-Tau (217P) Plasma | Positive (pTau217 > 0.378 pg/mL) | 285 | 322 | 88.5% (285/322) (84.9%, 91.4%) | 33.6% (322/959) | 11.82 (8.65, 16.25) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 72 | 191 | 37.7% (72/191) (317%, 44.0%) | 19.9% (191/959) | 0.93 (0.71, 1.20) | | | Negative (pTau217 ≤0.223 pg/mL) | 21 | 445 | 4.7% (21/445) (3.2%, 6.9%) | 46.5% (445/959) | 0.08 (0.05, 0.11) | | | Total | 378 | 958 | Prevalence of amyloid positivity= 39.5% | | | | *95%CIs are calculated based on 95%CIs of corresponding likelihood ratios **95%CIs are calculated using an asymptotic method for ratios of two independent binomial proportions | | | | | | | Of the 958 evaluable subjects, 378 had amyloid PET scan positive results and 580 had amyloid negative PET scan result. Of the 378 subjects with an amyloid PET scan positive result, 285 subjects had a positive Elecsys Phospho-Tau (217P) Plasma result (positive percent agreement was 75.4% (285/378) with 95% CI: (70.8%, 79.5%). Of the 580 with an amyloid PET scan negative result, 424 subjects had a negative Elecsys Phospho-Tau (217P) Plasma result (negative percent agreement was 73.1% (424/580) with 95% CI: (69.4%, 76.6%). The prevalence of amyloid PET scan positivity was 39.5% (378/958) overall in the study population. K261686 - Page 27 of 37 {27} In the study, there were 322 out of 958 (33.6%) subjects with positive Elecsys Phospho-Tau (217P) Plasma results. Among them, 285 subjects had amyloid PET scan positive results (PV for positive test results was 88.5% (285/322) with 95%CI: (84.9%, 91.4%)). The positive Elecsys Phospho-Tau (217P) Plasma results indicate an increase of the likelihood of amyloid PET scan positive results (PV for subjects with positive Elecsys Phospho-Tau (217P) Plasma results (88.5%) is statistically higher than prevalence of 39.5%: the lower bound of 95% CI for the PV of 84.9% > prevalence of 39.5%). In the study, there were 445 out of 958 (46.5%) subjects with negative Elecsys Phospho-Tau (217P) Plasma results. Among them, 21 subjects had amyloid PET positive results (PV for negative test results was 4.7% (21/445) with 95% CI: (3.2%, 6.9%) and NPV=95.3% (424/445) with 95% CI: (93.1%, 96.8%)). The negative Elecsys Phospho-Tau (217P) Plasma results indicate a low likelihood of the amyloid PET scan positive results (PV for subjects with negative Elecsys Phospho-Tau (217P) Plasma results (4.7%) is statistically lower than prevalence of 39.5%: the upper bound of 95% CI for the PV of 6.9% < prevalence of 39.5%). In the study, there were 191 out 958 (19.9%) subjects with intermediate Elecsys Phospho-Tau (217P) Plasma results. Of the 191 subjects with an intermediate Elecsys Phospho-Tau (217P) Plasma result, 72 had amyloid PET positive results. The PV for intermediate Elecsys Phospho-Tau (217P) Plasma results was 37.7% (72/191) with 95% CI: (31.7%, 44.0%). In conclusion, the data of clinical performance study support that positive Elecsys Phospho-Tau (217P) Plasma results are consistent with amyloid PET scan positive results and Elecsys Phospho-Tau (217P) Plasma negative results are consistent with amyloid PET scan negative results. ### 2) Sub-group Analysis #### a. MMSE The clinical performance measures of the Elecsys Phospho-Tau (2171P) Plasma assay are stratified by MMSE subgroups and summarized in the table below. | MMSE | Test Result | Amyloid PET Visual | | Predictive Value % (n/N) (95% CI*) | Frequency of Results % (n/N) | Likelihood Ratio (95% CI**) | | --- | --- | --- | --- | --- | --- | --- | | | | Positive | N | | | | | MMSE 28–30 | Positive (pTau217 > 0.378 pg/mL) | 51 | 66 | 77.3% (67.3%, 85.0%) | 22.4% (66/295) | 9.46 (5.72, 15.80) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 21 | 78 | 26.9% (19.2%, 35.7%) | 26.4% (78/295) | 1.02 (0.66, 1.54) | | | Negative (pTau217 ≤0.223 pg/mL) | 6 | 151 | 4.0% (1.9%, 7.9%) | 51.2% (151/295) | 0.12 (0.05, 0.24) | | | Total | 78 | 295 | Prevalence of amyloid positivity= 26.4% | | | | MMSE 25–27 | Positive (pTau217 > 0.378 pg/mL) | 103 | 114 | 90.4% (84.2%, 94.4%) | 31.4 (114/363) | 14.74 (8.36, 26.40) | K261686 - Page 28 of 37 {28} | MMSE | Test Result | Amyloid PET Visual | | Predictive Value % (n/N) (95% CI*) | Frequency of Results % (n/N) | Likelihood Ratio (95% CI**) | | --- | --- | --- | --- | --- | --- | --- | | | | Positive | N | | | | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 31 | 76 | 40.8% (31.4%, 50.7%) | 20.9% (76/363) | 1.08 (0.72, 1.62) | | | Negative (pTau217 ≤0.223 pg/mL) | 7 | 173 | 4.0% (2.0%, 7.8%) | 47.7% (173/363) | 0.07 (0.03, 0.13) | | | **Total** | 141 | 363 | *Prevalence of amyloid positivity= 38.8%* | | | | **MMSE 22–24** | Positive (pTau217 > 0.378 pg/mL) | 84 | 91 | 92.3% (85.7%, 96.1%) | 40.1% (91/227) | 13.70 (6.86, 28.16) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 15 | 30 | 50.0% (34.2%, 65.8%) | 13.2% (30/227) | 1.14 (0.59, 2.20) | | | Negative (pTau217 ≤0.223 pg/mL) | 7 | 106 | 6.6% (3.3%, 12.3%) | 46.7% (106/227) | 0.08 (0.04, 0.16) | | | **Total** | 106 | 227 | *Prevalence of amyloid positivity=46.7%* | | | | **MMSE 21 and 22** | Positive (pTau217 > 0.378 pg/mL) | 47 | 51 | 92.2% (84.8%, 96.7%) | 69.9% (51/73) | 4.43 (2.11, 11.04) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 5 | 7 | 71.4% (38.4%, 91.5%) | 9.6% (7/73) | 0.94 (0.24, 4.07) | | | Negative (pTau217 ≤0.223 pg/mL) | 1 | 15 | 6.7% (1.2%, 27.9%) | 20.5% (15/73) | 0.03 (0.00, 0.15) | | | **Total** | 53 | 73 | *Prevalence of amyloid positivity=72.6%* | | | | *95%CIs are calculated based on 95%CIs of corresponding likelihood ratios **95%CIs are calculated using an asymptotic method for ratios of two independent binomial proportions | | | | | | | ### b. Sex The clinical performance measures of the Elecsys Phospho-Tau (217P) Plasma assay are stratified by sex and summarized in the table below. | Sex | Test Result | Amyloid PET Visual | | Predictive Value % (n/N) (95% CI*) | Frequency of Results % (n/N) | Likelihood Ratio (95% CI**) | | --- | --- | --- | --- | --- | --- | --- | | | | Positive (n) | N | | | | | **Male** | Positive (pTau217 > 0.378 pg/mL) | 122 | 144 | 84.7% (122/144) (78.8%, 89.3%) | 33.4% (144/431) | 8.19 (5.48, 2.39) | K261686 - Page 29 of 37 {29} | Sex | Test Result | Amyloid PET Visual | | Predictive Value % (n/N) (95% CI*) | Frequency of Results % (n/N) | Likelihood Ratio (95% CI**) | | --- | --- | --- | --- | --- | --- | --- | | | | Positive (n) | N | | | | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 40 | 104 | 38.5% (40/104) (30.6%, 46.7%) | 24.1% (104/431) | 0.92 (0.65, 1.30) | | | Negative (pTau217 ≤0.223 pg/mL) | 12 | 183 | 6.6% (12/183) (3.9%, 10.7%) | 42.5% (183/431) | 0.10 (0.06, 0.18) | | | **Total** | 174 | 431 | *Prevalence of amyloid positivity=40.4%* | | | | **Female** | Positive (pTau217 > 0.378 pg/mL) | 163 | 178 | 91.6% (163/178) (87.0%, 94.7%) | 33.8% (178/527) | 17.21 (10.57, 28.32) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 32 | 87 | 36.8% (32/87) (28.1%, 46.3%) | 16.5% (87/527) | 0.92 (0.62, 1.36) | | | Negative (pTau217 ≤0.223 pg/mL) | 9 | 262 | 3.4% (9/262) (1.8%, 6.2%) | 49.7% (262/527) | 0.06 (0.03, 0.10) | | | **Total** | 204 | 527 | *Prevalence of amyloid positivity=38.7%* | | | | *95%CIs are calculated based on 95%CIs of corresponding likelihood ratios **95%CIs are calculated using an asymptotic method for ratios of two independent binomial proportions | | | | | | | # c. Age The clinical performance measures of the Elecsys Phospho-Tau (217P) Plasma assay are stratified by age groups and summarized in the table below. | Age | Test Result | Amyloid PET Visual | | Predictive Value % (n/N) (95% CI*) | Frequency of Results % (n/N) | Likelihood Ratio (95% CI**) | | --- | --- | --- | --- | --- | --- | --- | | | | Positive (n) | N | | | | | **55-70 yrs** | Positive (pTau217 > 0.378 pg/mL) | 76 | 88 | 86.4% (76/88) (78.4%, 91.8%) | 20.5% (88/430) | 18.88 (10.84, 33.21) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 25 | 84 | 29.8% (25/84) (21.8%, 38.8%) | 19.5% (84/430) | 1.26 (0.83, 1.89) | | | Negative (pTau217 ≤0.223 pg/mL) | 7 | 258 | 2.7% (7/258) (1.3%, 5.2%) | 60.0% (258/430) | 0.08 (0.04, 0.16) | | | **Total** | 108 | 430 | *Prevalence of amyloid positivity=25.1%* | | | | **71-85 yrs** | Positive (pTau217 > 0.378 pg/mL) | 209 | 234 | 89.3% (209/234) (85.3%, 92.4%) | 44.3% (234/528) | 7.99 (5.53, 11.70) | K261686 - Page 30 of 37 {30} | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 47 | 107 | 43.9% (47/107) (35.8%, 52.4%) | 20.3% (107/528) | 0.75 (0.53, 1.05) | | --- | --- | --- | --- | --- | --- | --- | | | Negative (pTau217 ≤0.223 pg/mL) | 14 | 187 | 7.5% (14/187) (4.6%, 11.8%) | 35.4% (187/528) | 0.08 (0.05, 0.13) | | | **Total** | 270 | 528 | *Prevalence of amyloid positivity=51.1%* | | | | *95%CIs are calculated based on 95%CIs of corresponding likelihood ratios **95%CIs are calculated using an asymptotic method for ratios of two independent binomial proportions | | | | | | | # d. Race The clinical performance measures of the Elecsys Phospho-Tau (2171P) Plasma assay are stratified by race and summarized in the table below. | Race | Test Result | Amyloid PET Visual | | Predictive Value % (n/N) (95% CI*) | Frequency of Results % (n/N) | Likelihood Ratio (95% CI**) | | --- | --- | --- | --- | --- | --- | --- | | | | Positive (n) | N | | | | | **White** | Positive (pTau217 > 0.378 pg/mL) | 276 | 307 | 89.9% (276/307) (86.4%, 92.6%) | 37.9% (307/811) | 10.99 (7.83, 15.53) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 69 | 166 | 41.6% (69/166) (35.0%, 48.3%) | 20.5% (166/811) | 0.88 (0.67, 1.15) | | | Negative (pTau217 ≤0.223 pg/mL) | 18 | 338 | 5.3% (18/338) (3.4%, 8.1%) | 41.7% (338/811) | 0.07 (0.04, 0.11) | | | **Total** | 363 | 811 | *Prevalence of amyloid positivity=44.8%* | | | | **African American** | Positive (pTau217 > 0.378 pg/mL) | 6 | 11 | 54.5% (6/11) (30.2%, 75.7%) | 10.0% (11/110) | 10.80 (3.89, 28.05) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 2 | 19 | 10.5% (2/19) (3.1%, 5.9%) | 17.3% (19/110) | 1.06 (0.28, 3.15) | | | Negative (pTau217 ≤0.223 pg/mL) | 3 | 80 | 3.8% (3/80) (1.4%, 7.6%) | 72.7% (80/110) | 0.35 (0.12, 0.74) | | | **Total** | 11 | 110 | *Prevalence of amyloid positivity=10.0%* | | | | **Asian** | Positive (pTau217 > 0.378 pg/mL) | 1 | 1 | 100.0% (1/1) (21.7%, 100.0%) | 6.7% (1/15) | Inf (1.81, inf) | | | Intermediate (0.223< pTau217 ≤ 0.378 pg/mL) | 1 | 4 | 25.0% (…