EUROIMMUN ANTI-PLA2R IFA

• K Number: DEN140002
• Device Name: EUROIMMUN ANTI-PLA2R IFA
• Manufacturer: EUROIMMUN US
• Date Cleared: 2014-05-29 ( 62 days)
• Product Code: PGV
• Regulation Number: 866.5780
• Type: Post-NSE
• Panel: IM
• Reference & Predicate Devices: N/A

Intended Use

The EUROIMMUN Anti-PLA2R IFA is intended for the qualitative determination of IgG class autoantibodies against phospholipase A2 receptor (PLA2R) in human serum. It is used as an aid in the diagnosis of primary membranous glomerulonephritis (pMGN), in conjunction with other laboratory and clinical findings.

Device Description

The kit (for 50 determinations) contains an instruction booklet and the following materials:

• . Slides, each containing 5x2 BIOCHIPs coated with PLA2R-transfected and controltransfected cells
• Fluorescein-labelled anti-human IgG (goat), ready for use (1 x 1.5 ml);
• Positive control: anti-PLA2R, ready for use (1 x 0.1 ml) ●
• . Negative control: autoantibody negative, ready for use (1 x 0.1 ml)
• Salt for PBS pH 7.2 (x2)
• Tween 20 (2 x 2.0 ml) .
• Embedding medium, ready for use (1 x 3.0 ml) •
• . Cover glasses (62 mm x 23 mm) (x12)

The EUROIMMUN IFA is an assay for standardized detection of anti-PLA2R antibodies utilized in laboratories familiar with indirect immunofluorescence. The test kit consists of slides, which contain BIOCHIPs coated with PLA2R transfected cells and control-transfected cells, fluorescein-labelled anti-human IgG (goat), a positive control for anti-PLA2R, a negative control. a salt for preparation of PBS. Tween 20, embedding medium, cover glasses and an instruction booklet.

AI/ML Overview

This document describes the acceptance criteria and the studies performed to evaluate the performance of the EUROIMMUN Anti-PLA2R IFA for the qualitative determination of IgG class autoantibodies against phospholipase A2 receptor (PLA2R) in human serum, as an aid in the diagnosis of primary membranous glomerulonephritis (pMGN).

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for analytical performance validation focused on qualitative results and fluorescence intensity deviation. For clinical performance, the acceptance criteria are implied by the reported clinical sensitivity and specificity for pMGN and control groups.

Performance Characteristic	Acceptance Criteria	Reported Device Performance
Analytical Performance
Precision/Reproducibility	All qualitative results of positive samples be positive and not exceed the acceptable deviation of fluorescence intensity of ±1 intensity level, and results of negative samples be negative.	Intra-assay: Samples 1 & 2 (Negative): 100% negative (0% positive). Samples 3-6 (Positive): 100% positive. Mean intensities are 0, 0, 1.8, 2.4, 3, 3.9 which would provide qualitative results of Negative, Negative, Positive, Positive, Positive, Positive. This meets the criteria of all positive samples being positive and negative samples being negative. While intensity deviation for positive samples isn't explicitly stated for individual runs, the overall qualitative outcome alignment suggests adherence.
Inter-assay: Samples 1 & 2 (Negative): 100% negative (0% positive). Samples 3-6 (Positive): 100% positive. Mean intensities are 0, 0, 1, 2, 3, 4 which would provide qualitative results of Negative, Negative, Positive, Positive, Positive, Positive. This meets the criteria.
Lot-to-lot: Variation in fluorescence intensity was within ±1 intensity level unit for positives. All negative samples were tested as negative with intensity level at 0. This meets the criteria.
Site-to-site: Samples 1 & 2 (Negative): 100% negative (0% positive). Samples 3-7 (Positive): 100% positive. Mean intensities are 0, 0, 1, 1.95, 3, 4, 3.5 which would provide qualitative results of Negative, Negative, Positive, Positive, Positive, Positive, Positive. This meets the criteria.
Stability (Kit)	Results not to exceed ±1 intensity level deviation compared to day 0 for real-time; no deviation in fluorescence intensity level exceeding ±1 for accelerated.	Real-time: Results were compared to day 0 and did not exceed ±1. Supports 18 months stability.
Accelerated: Results showed no deviation in fluorescence intensity level exceeding ±1. Supports 18 months stability at 2-8°C.
Analytical Specificity (Interference)	Deviation in fluorescence intensity level not to exceed ±1.	No interference was observed for concentrations up to 500 mg/dL for hemoglobin, 2000 mg/dL for triglyceride and 40 mg/dL for bilirubin. This meets the criteria.
Analytical Specificity (Cross-reactivity)	Implied: No cross-reactivity with other autoimmune diseases.	All 60 sera from clinically characterized conditions (thyroiditis, SLE, SSc, RA, cANCA, pANCA, Goodpasture Syndrome, HBsAg) were negative, indicating no cross-reactivity.
Assay Cut-off Validation	Implied: Appropriate differentiation between pMGN and control groups.	At 1:10 dilution: 23 out of 35 pMGN samples (65.7%) were positive. All non-membranous glomerulonephritis (n=182) and normal healthy blood donors (n=150) were negative. The 1:10 dilution was determined as the cut-off.
Clinical Performance
Clinical Sensitivity	Implied: Sufficiently high for an aid in diagnosis.	77.1% (95% CI: 71.7 - 81.9%) for pMGN patients (N=275).
Clinical Specificity	Implied: Sufficiently high to differentiate from other conditions.	100.0% (95% CI: 98.7 - 100.0%) for a total of 285 control samples from sMGN, non-membranous glomerulonephritis, SLE, SSc, PSA, RA, and Thyroiditis groups. Breakdown: sMGN (N=68): 100.0%; Non-membranous glomerulonephritis (N=63): 100.0%; SLE (N=30): 100.0%; SSc (N=30): 100.0%; PSA (N=30): 100.0%; RA (N=14): 100.0%; Thyroiditis (N=50): 100.0%.

The reported device performance generally met or exceeded the implied acceptance criteria for analytical and clinical performance. The consistent qualitative results and controlled fluorescence intensity deviations in precision studies, along with the high specificity and reasonable sensitivity in clinical studies, support the device's intended use.

2. Sample sizes used for the test set and data provenance

• Analytical Performance Test Sets:

  • Intra-assay reproducibility: 6 characterized positive and negative serum samples, with 10 determinations each.
  • Inter-assay reproducibility: 6 characterized positive and negative serum samples, with 10 determinations each using two slides per run at five different times.
  • Lot-to-lot reproducibility: 2 sets of samples (6 samples per set: 2 negatives, 4 positives; and 2 negatives, 2 positives), tested with 3 different slide lots and 3 reagent lots respectively.
  • Site-to-site reproducibility: 7 characterized positive and negative serum samples, tested at 3 different sites, each with 2 runs per day for 5 days.
  • Interference: 3 serum samples (intensities 0, 1, 3), tested with 3 different concentrations of each interfering substance.
  • Cross-reactivity: Panel of 60 clinically characterized sera.
  • Assay Cut-off: 35 biopsy-proven pMGN patients, 182 non-membranous glomerulonephritis patients, and 150 normal healthy blood donors.

• Clinical Performance Test Set: 560 clinically characterized samples (275 from pMGN patients, 285 from control groups including sMGN, non-membranous glomerulonephritis, SLE, SSc, PSA, RA, Thyroiditis).

• Expected Values/Reference Range: 128 samples from normal healthy European population and 189 samples from normal healthy US population.

• Data Provenance: The document does not explicitly state the country of origin for all samples. However, for the reference range, it specifies "normal healthy European population" and "normal healthy US population." The clinical samples are described as "clinically characterized samples," often associated with patient cohorts from studies, which could be retrospective or prospective. The context of "samples drawn within 8 weeks after biopsy, before treatment" for pMGN patients and exclusion of "patients who had been or were currently being treated with immunosuppressive drugs" suggests retrospective collection from specific patient groups that fit these criteria at the time of collection.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

• Analytical Performance: For site-to-site reproducibility, "each result was evaluated by two different technicians independently." Qualifications for these technicians are not provided.
• Clinical Performance:
  • Ground Truth for pMGN: "pMGN diagnosis was based on renal biopsy and was considered to be idiopathic/primary when no secondary cause of MGN was suspected on the basis of clinical and laboratory criteria." The number and qualifications of experts (e.g., pathologists, nephrologists) who rendered these diagnoses are not specified.
  • Ground Truth for sMGN: "sMGN diagnosis was based on serological tests and clinical evaluation." The number and qualifications of experts are not specified.
  • Ground Truth for other control groups: Diagnosis was based on established diagnostic criteria and clinical evaluation. The number and qualifications of experts are not specified.
• Assay Cut-off: Ground truth was based on "biopsy proven pMGN patients," "non-membranous glomerulonephritis patients," and "normal healthy blood donors," implying clinical and pathological diagnoses, but expert details are not specified.

4. Adjudication method for the test set

• Analytical Performance: For site-to-site reproducibility, "Each result was evaluated by two different technicians independently." This implies a form of independent double-reading. However, explicit adjudication rules (e.g., how discrepancies were resolved, 2+1, 3+1, none) are not provided.
• Clinical Performance: No explicit adjudication method is described for establishing the clinical ground truth diagnoses (e.g., for pMGN, sMGN, or control groups). The diagnoses were established based on biopsy, serological tests, and clinical evaluation, but there is no mention of blinded review or consensus processes by multiple experts for the clinical ground truth.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

• A Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This device is an Indirect Immunofluorescent Antibody Assay (IFA), which is a diagnostic laboratory test that relies on laboratory technicians for interpretation of fluorescence intensity under a microscope, rather than an AI system assisting human readers. The context provided does not involve AI assistance.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done

• This is not an algorithm-only device. It is an IFA kit for laboratory use. While internal scoring of fluorescence intensity is mentioned, the evaluation of fluorescence and subsequent qualitative or quantitative reporting is performed by laboratory technicians using a fluorescent microscope. The device itself does not perform automated image analysis or diagnosis without human intervention.

7. The type of ground truth used

• Analytical Performance: The ground truth for analytical performance studies generally relied on pre-characterized serum samples (e.g., known positive or negative, with defined fluorescence intensities).
• Assay Cut-off Validation: Ground truth was primarily based on histopathological diagnosis (renal biopsy) for pMGN, and clinical diagnosis with laboratory findings for non-membranous glomerulonephritis and normal healthy individuals.
• Clinical Performance:
  • Primary Membranous Glomerulonephritis (pMGN): Diagnosis was primarily based on renal biopsy findings, supplemented by clinical and laboratory criteria to rule out secondary causes. This is a form of pathology-based ground truth combined with clinical evaluation.
  • Secondary Membranous Glomerulonephritis (sMGN): Diagnosis was based on serological tests and clinical evaluation.
  • Other Control Groups: Diagnoses were based on established diagnostic criteria and clinical evaluation.

8. The sample size for the training set

• No explicit training set is mentioned in the context of an algorithm or AI model. This device is a traditional IFA assay. The closest equivalent to "training" in this context would be the data used to validate the assay parameters, such as the initial establishment of the assay cut-off. For the assay cut-off, the sample size used was 35 pMGN patients, 182 non-membranous glomerulonephritis patients, and 150 normal healthy blood donors.

9. How the ground truth for the training set was established

• As there is no "training set" in the AI sense for this traditional IFA assay, the question refers to how ground truth was established for the samples used to determine the assay cut-off and validate its performance.
• Assay Cut-off Establishment:
  • pMGN samples: Ground truth was "biopsy proven pMGN patients." This refers to a pathology-based diagnosis established by medical professionals.
  • Non-membranous glomerulonephritis patients: Ground truth was their clinical diagnosis.
  • Normal healthy blood donors: Ground truth was their healthy status confirmed by medical screening, not having the disease markers.

Regulation Number and Section

§ 866.5780 Anti-phospholipase A2 receptor immunological test system.

(a)
Identification. An anti-phospholipase A2 receptor immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies in human blood samples that react with phospholipase A2 receptor. The measurements aid in the diagnosis of primary membranous glomerulonephritis (pMGN), in conjunction with other laboratory and clinical findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation must include:
(i) A detailed description of the device that includes:
(A) A detailed description of all parts of the test system, including a description of the assay parts in the kit and all required ancillary reagents;
(B) A detailed description of instrumentation and equipment, and illustrations or photographs of non-standard equipment or methods if applicable;
(C) Detailed documentation of the device software, including standalone software applications and hardware-based devices that incorporate software where applicable;
(D) A detailed description of appropriate internal and external quality controls that are recommended or provided. The description must identify those control elements that are incorporated into the recommended testing procedures;
(E) Detailed specifications for sample collection, processing, and storage;
(F) A detailed description of methodology and assay procedure; and
(G) Detailed specification of the criteria for test results interpretation and reporting.
(ii) Information that demonstrates the performance characteristics of the device, including:
(A) Device precision/reproducibility data generated from within-run, between-run, between-day, between-lot, between-operator, between-instruments, between-site, and total precision for multiple nonconsecutive days as applicable. A well-characterized panel of patient samples or pools from the intended use population that covers the device measuring range must be used;
(B) Device linearity data generated from patient samples covering the assay measuring range if applicable;
(C) Information on traceability to a reference material and description of value assignment of calibrators and controls if applicable;
(D) Device analytical sensitivity data, including limit of blank, limit of detection, and limit of quantitation if applicable;
(E) Device analytical specificity data, including interference by endogenous and exogenous substances, as well as cross-reactivity with samples derived from patients with other autoimmune diseases or conditions;
(F) Device instrument carryover data when applicable;
(G) Device stability data including real-time stability under various storage times and temperatures;
(H) Specimen stability data including stability under various storage times, temperatures, freeze-thaw, and transport conditions where appropriate;
(I) Method comparison data generated by comparison of the results obtained with the device to those obtained with a legally marketed predicate device with similar indication of use. Patient samples from the intended use population covering the device measuring range must be used;
(J) Specimen matrix comparison data if more than one specimen type or anticoagulant can be tested with the device. Samples used for comparison must be from patient samples covering the device measuring range;
(K) A description of how the assay cut-off (the medical decision point between positive and negative) was established and validated as well as supporting data;
(L) A clinical performance assessment established by comparing data generated by testing samples from the intended use population and differential diagnosis groups with the device to the clinical diagnostic standard. Diagnosis of pMGN must be based primarily on clinical history, physical examination, laboratory tests (including urinalysis), and renal biopsy. Membranous glomerulonephritis is considered to be idiopathic/primary when no secondary cause can be elucidated on the basis of clinical and laboratory criteria. The differential diagnosis groups must include secondary membranous glomerulonephritis, membranoproliferative glomerulonephritis, lupus nephritis, focal segmental glomerulosclerosis, IgA nephritis, diabetic nephropathy, systemic lupus erythematosus, systemic sclerosis, and Goodpasture syndrome. Diagnosis of autoimmune and immune-mediated diseases that are associated with membranous glomerulonephritis must be based on established diagnostic criteria and clinical evaluation. For all samples, clinical criteria, including demographic information, must be considered in the differentiation between pMGN and secondary membranous glomerulonephritis. The clinical validation results must demonstrate correlation clinical sensitivity and clinical specificity between the test values and the presence or absence of pMGN. The data must be summarized in tabular format comparing the interpretation of results to the disease status; and
(M) Expected/reference values generated by testing an adequate number of samples from apparently healthy normal individuals.
(iii) Identification of risk mitigation elements used by the device, including a description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.
(2) The label required under § 809.10(a) and labeling required under § 809.10(b) of this chapter must include warnings relevant to the assay including:
(i) A warning statement that explains: The device is for use by laboratory professionals in a clinical laboratory setting;
(ii) A warning statement that explains: The test is not a standalone test but an adjunct to other clinical information. A diagnosis of pMGN or secondary MGN should not be made based on a single test result. The clinical symptoms, results on physical examination, and laboratory tests (
e.g., serological tests), when appropriate, should always be taken into account when considering the diagnosis of primary versus secondary MGN;(iii) A warning statement that explains: Absence of circulating PLA2R autoantibody does not rule out a diagnosis of pMGN; and
(iv) A warning statement that explains: The assay has not been demonstrated to be effective for monitoring the stage of disease or its response to treatment.
(3) The labeling required under § 809.10(b) of this chapter must include a description of the protocol and performance studies performed in accordance with paragraph (b)(1)(ii) of this section and a summary of the results.