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Amphetamines II (AMPII) is an in vitro diagnostic test for the qualitative and semiquantitative detection of amphetamines and methamphetamines in human urine on COBAS INTEGRA systems at cutoff concentrations of 300 ng/mL, 500 ng/mL and 1000 ng/mL when calibrated with a-methamphetamine. Semiquantitative test results may be obtained that permit laboratories to assess assay performance as part of a quality control program. Semiquantitative assays are intended to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas chromatography/mass spectrometry (GC/MS).
Amphetamines II provides only a preliminary analytical test result. A more specific alternate chemical method must be used in order to obtain a confirmed analytical result. Gas chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method.1 Clinical consideration and professional judgement should be applied to any drug of abuse test result, particularly when preliminary positive results are used.
The Amphetamines II test is an immunoassay for use on automated clinical chemistry analyzers. The device consists of two wet reagents; a soluble drug-conjugate, and an antibody-bound microparticle solution. During the assay, in the absence of sample drug in urine, soluble drug-conjugates bind to antibody-bound microparticles, causing the formation of particle aggregates. When a urine sample contains the drug in question, this drug competes with the drug derivative conjugate for microparticle-bound antibody. Antibody bound to sample drug is no longer available to promote particle aggregation, and subsequent particle lattice formation is inhibited. The rate of absorbance change is proportional to the concentration of drug in the sample. Calibrators, ranging in concentration from 0-5000 ng/mL depending on cutoff and test mode, are run with the assay. Concentrations of controls and unknowns are calculated from the standard curve in semi-quantitative mode. Results for controls or calibrators are determined as preliminary positive or negative relative to the cutoff in qualitative mode.
C.f.a.s. DAT Qualitative Clinical, C.f.a.s. DAT Qualitative Plus, C.f.a.s. DAT Qualitative Plus Clinical, Preciset DAT Plus I Calibrators, and Preciset DAT Plus II Calibrators are ready to use, multianalyte calibrators prepared by the quantitative addition of drug or drug metabolite to drug-free human urine.
Control Set DAT I, II, and III, and Control Set DAT Clinical are ready to use multianalyte controls prepared by the quantitative addition of drug or drug metabolite to drug-free urine.
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The provided document describes the Amphetamines II Assay for Integra Family of Analyzers, an in vitro diagnostic test for the qualitative and semiquantitative detection of amphetamines and methamphetamines in human urine. The device's performance is primarily evaluated through precision and accuracy studies against Gas Chromatography/Mass Spectrometry (GC/MS).
1. Table of Acceptance Criteria and Reported Device Performance
The general acceptance criteria for this type of immunoassay are implicit in the performance studies: accurate detection of amphetamines/methamphetamines in urine relative to established cutoffs, with good precision and minimal interference from other substances. The detailed criteria would typically be established internally by the manufacturer and submitted to regulatory bodies.
Here's a summary of the stated performance, interpreted against general expectations for such assays:
| Acceptance Criterion (Implicit) | Reported Device Performance |
|---|---|
| Precision | Qualitative Mode: |
- 300 ng/mL Cutoff (MAMP): 84/84 Negative at zero, -75%, -50% of cutoff. 83/84 Negative at -25% of cutoff. 27/84 Negative, 57/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.
- 500 ng/mL Cutoff (MAMP): 84/84 Negative at zero, -75%, -50% of cutoff. 84/84 Negative at -25% of cutoff. 28/84 Negative, 56/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.
- 1000 ng/mL Cutoff (MAMP): 84/84 Negative at zero, -75%, -50%, -25% of cutoff. 13/84 Negative, 71/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.
- 300 ng/mL Cutoff (AMP): 84/84 Negative at zero, -75%, -50% of cutoff. 83/84 Negative at -25% of cutoff. 2/84 Negative, 82/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.
- 500 ng/mL Cutoff (AMP): 84/84 Negative at zero, -75%, -50% of cutoff. 82/84 Negative, 2/84 Positive at -25% of cutoff. 6/84 Negative, 78/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.
- 1000 ng/mL Cutoff (AMP): 84/84 Negative at zero, -75%, -50% of cutoff. 82/84 Negative, 2/84 Positive at -25% of cutoff. 7/84 Negative, 77/84 Positive at cutoff. 1/84 Negative, 83/84 Positive at +25% of cutoff. 84/84 Positive at +50%, +75%, +100% of cutoff.
Semiquantitative Mode (SD, CV% ranges reported):
- For MAMP at 300, 500, 1000 ng/mL cutoffs:
- Repeatability (within-run) CV% for zero to +100% of cutoff generally good (e.g., 5.7%-95.4% for 300 ng/mL, with highest CVs at very low concentrations).
- Intermediate Precision (total) CV% for zero to +100% of cutoff also generally good (e.g., 6.5%-123.8% for 300 ng/mL, with highest CVs at very low concentrations).
- For AMP at 300, 500, 1000 ng/mL cutoffs:
- Repeatability (within-run) CV% generally good (e.g., 5.3%-110.0% for 300 ng/mL).
- Intermediate Precision (total) CV% also generally good (e.g., 6.5%-135.7% for 300 ng/mL). |
| Accuracy (Agreement with GC/MS) | MAMP (d-methamphetamine):
- Qualitative & Semiquantitative Assays:
- 100% agreement for High Positive by GC/MS (greater than +50% of cutoff) across all cutoffs (300, 500, 1000 ng/mL).
- 100% agreement for Near Cutoff Positive by GC/MS (between cutoff and +50%) across all cutoffs.
- For Low Negatives (below cutoff) / Near Cutoff Negative (between -50% and cutoff):
- 300 ng/mL Cutoff: 90% (Qualitative & Semiquantitative).
- 500 ng/mL Cutoff: 91% (Qualitative & Semiquantitative).
- 1000 ng/mL Cutoff: 90% (Qualitative & Semiquantitative).
AMP (d-amphetamine):
- Qualitative & Semiquantitative Assays:
- 100% agreement for High Positive by GC/MS (greater than +50% of cutoff) across all cutoffs.
- 100% agreement for Near Cutoff Positive by GC/MS (between cutoff and +50%) across all cutoffs.
- For Low Negatives (below cutoff) / Near Cutoff Negative (between -50% and cutoff):
- 300 ng/mL Cutoff: 92.5% (Qualitative & Semiquantitative).
- 500 ng/mL Cutoff: 92.5% (Qualitative & Semiquantitative).
- 1000 ng/mL Cutoff: 97.5% (Qualitative), 95% (Semiquantitative). |
| Analytical Specificity / Cross-reactivity | A wide range of structurally similar compounds (e.g., MDMA, MDA, MDEA, BDB, l-Methamphetamine, l-Amphetamine) show varying degrees of cross-reactivity, with some "designer drugs" showing higher cross-reactivity than others (e.g., MDMA at 264% at 300 ng/mL MAMP equivalent). Most other common drugs/substances (e.g., acetaminophen, ibuprofen, caffeine, opiates, benzodiazepines) show either no significant interference or proper recovery of spiked samples at specified high concentrations (100,000 ng/mL). |
| Interference from Common Substances | No interference observed from high concentrations of common urine components (e.g., Acetone, Ascorbic Acid, Bilirubin, Creatinine, Ethanol, Glucose, Hemoglobin, Human serum albumin, Oxalic Acid, Sodium Chloride, Urea) for both qualitative and semiquantitative modes across all cutoffs when spiked with d-methamphetamine or d-amphetamine at -25% and +25% of cutoff. |
| pH and Specific Gravity Interference | No control cross-overs for specific gravities from 1.001 to 1.034. ≤ 5% cross-overs for pH ranging from 4.5 to 8.0. |
2. Sample Size Used for the Test Set and Data Provenance
- Precision Test Set Sample Size:
- For each cutoff (300, 500, 1000 ng/mL) and each drug (d-methamphetamine, d-amphetamine): 9 concentrations were tested.
- Each concentration was tested in 2 replicates per run, 2 runs per day for 21 days.
- Total N = 84 determinations per concentration level. (e.g., 9 concentrations * 84 determinations = 756 total determinations per drug per cutoff for precision).
- Data Provenance: "9 samples obtained from a human urine sample pool" for initial spiking. The source of this urine pool is not specified (e.g., country of origin). This was a retrospective study measuring inherent characteristics of the assay.
- Accuracy Test Set Sample Size:
- Negative Samples: 36 urine samples confirmed negative by GC/MS.
- Positive Samples: 36 samples confirmed positive by GC/MS (containing d-methamphetamine or d-amphetamine).
- Near Cutoff Samples: For each cutoff, 4 negative near cutoff samples and 4 positive near cutoff samples were assayed.
- 4 additional unaltered samples (d-methamphetamine, 85-204 ng/mL) evaluated with 500 ng/mL cutoff.
- Data Provenance: "samples obtained from a clinical laboratory" and "unaltered near cutoff samples." Geographic origin is not specified. This was a retrospective study comparing the device to a gold standard.
- Analytical Specificity Test Set Sample Size: Not explicitly stated as a count of individual samples, but inhibition curves were generated for each of the many tested compounds.
- Interference Test Set Sample Size: Not explicitly stated as a count of individual samples, but various compounds were added to urine pools at specified concentrations. This appears to be a laboratory retrospective study.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications
- The ground truth for the test set (accuracy study) was established by Gas Chromatography/Mass Spectrometry (GC/MS).
- This method is explicitly stated as "the preferred confirmatory method" and the device's traceability is stated as "Traceability: This method has been standardized against GC/MS."
- There is no mention of human expert involvement in establishing the ground truth; it relies on the analytical capabilities of GC/MS. Therefore, qualifications of human experts for ground truth are not applicable in this context.
4. Adjudication Method for the Test Set
- Adjudication methods like 2+1 or 3+1 typically apply to studies where human readers interpret data, and discrepancies need to be resolved.
- In this context, the device's results are compared directly to the quantitative results from GC/MS. There is no human interpretation of the device's output or a need for expert adjudication of "truth" beyond the GC/MS result itself. Therefore, no adjudication method as described (e.g., 2+1) was used or is applicable.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done
- No, an MRMC comparative effectiveness study was not done.
- This device is an automated immunoassay for analytical detection, not a system designed to assist human readers in a diagnostic task (e.g., interpreting medical images). Therefore, a study comparing human readers with and without AI assistance is not relevant to this device.
6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) Was Done
- Yes, this entire submission details standalone (algorithm only) performance.
- The Amphetamines II Assay is an automated in vitro diagnostic test run on COBAS INTEGRA systems. Its performance data (precision, accuracy, specificity, interference) are all based on the analytical capabilities of the instrument and reagents without human intervention in the result determination process. Human involvement would be limited to operating the analyzer, reviewing quality control, and interpreting the final preliminary positive/negative result in a clinical context (which the device explicitly states should be followed by GC/MS confirmation and professional judgment).
7. The Type of Ground Truth Used
- The primary ground truth used for the accuracy study was Gas Chromatography/Mass Spectrometry (GC/MS) results. This is a highly accurate and specific analytical method, often considered the "gold standard" for drug confirmation in toxicology.
- For precision and some interference studies, spiked urine samples with known concentrations of d-methamphetamine or d-amphetamine were used, where the known concentration is the ground truth.
8. The Sample Size for the Training Set
- The document does not mention a training set sample size.
- This is an immunoassay, not a machine learning or AI-based diagnostic tool that typically requires a large training set. The "training" of such a system involves the development and optimization of the reagents and assay parameters by the manufacturer, rather than a data-driven training process in the sense of machine learning.
9. How the Ground Truth for the Training Set Was Established
- As a traditional immunoassay, there is no explicit "training set" in the machine learning sense.
- The development of the assay's reagents, antibody specificity, and reaction conditions would have been guided by extensive biochemical research and experimentation to ensure specific binding and accurate signal generation across the desired concentration range. The "ground truth" during this development (or "training") phase would be the known concentrations of target analytes and interfering substances used to optimize the assay's performance characteristics. This is an iterative process of development and validation, rather than a single training set with established ground truth.
§ 862.3100 Amphetamine test system.
(a)
Identification. An amphetamine test system is a device intended to measure amphetamine, a central nervous system stimulating drug, in plasma and urine. Measurements obtained by this device are used in the diagnosis and treatment of amphetamine use or overdose and in monitoring levels of amphetamine to ensure appropriate therapy.(b)
Classification. Class II (special controls). An amphetamine test system is not exempt if it is intended for any use other than employment or insurance testing or is intended for Federal drug testing programs. The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9, provided the test system is intended for employment and insurance testing and includes a statement in the labeling that the device is intended solely for use in employment and insurance testing, and does not include devices intended for Federal drug testing programs (e.g., programs run by the Substance Abuse and Mental Health Services Administration (SAMHSA), the Department of Transportation (DOT), and the U.S. military).