K Number
K120986
Date Cleared
2012-08-16

(136 days)

Product Code
Regulation Number
866.3336
Reference & Predicate Devices
N/A
Predicate For
N/A
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended Use

The Focus Diagnostics' STRATIFY JCV® DxSelect™ assay is intended for the qualitative detection of antibodies to JC virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis patients receiving or considering natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only.

The assay is not intended for donor screening. The performance of this assay has not been established for use in other immunocompromised patient populations or patients with different disease conditions or undergoing other treatments or in neonates and pediatric patient populations.

Device Description

The Focus Diagnostics' STRATIFY JCV® DxSelect™ test is an ELISA assay. JC virus-like particles (VLP) are pre-coated onto 96-well microiter plates. Diluted serum or plasma specimens and controls are incubated in the wells to allow JCV-specific antibodies present in the specimens to react with the JC VLP antigen. Nonspecific reactants are removed by washing. Peroxidase-conjugated anti-human antibodies are added to react with JCV-specific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of optical density (OD). Specimen OD readings are compared with Cut-Off Calibrator OD readings to determine results. Each specimen result is reported as an index value. A specimen with an index value that is greater than a specified upper cut-off is reported as positive for detectable JCV-specific antibodies, whereas a specimen with an index value less than the specified lower cut-off is reported as negative for detectable JCV-specific antibodies. A specimen with an index value that is equal to or between the upper and lower cut-off values is reported as indeterminate. An indeterminate result requires further evaluation in the confirmation (inhibition) assay.

In the confirmation assay, soluble JC VLP antigen will compete with plate bound JC VLP antigen for the JCV-specific antibodies present in the serum or plasma specimens. After washing away the unbound antibodies, peroxidase-conjugated anti-human antibodies are added and bind to any captured JCVspecific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromagen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of OD. The percent inhibition is calculated to confirm presence of JCV-specific antibodies in the specimens with a percent inhibition value that is greater than the specified cut-off are reported as positive for detectable JCV-specific antibodies, whereas specimens with percent inhibition values less than or equal to the cut-off are reported as negative for detectable JCV-specific antibodies.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Acceptance Criteria and Device Performance for STRATIFY JCV® DxSelect™

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly present a "table of acceptance criteria" in the traditional sense, but rather demonstrates performance against the predicate device and established clinical guidelines. The key performance metrics evaluated are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) with a validated laboratory methodology (the predicate device) and JCV antibody positivity rate in certain patient populations.

MetricAcceptance Criteria (Implicit from Predicate & Clinical Relevance)Reported Device Performance
Agreement with Predicate (Pre-PML Samples)High positive agreement (especially for pre-PML samples, as JCV antibody positivity is a necessary step for PML development). The predicate device itself serves as the benchmark.100% Positive Agreement (31/31) with the validated assay (95% CI: 89.0% to 100%) for pre-PML samples.
Agreement with Predicate (Patients Receiving Natalizumab)High Positive Percent Agreement and Negative Percent Agreement with the validated laboratory methodology used in clinical studies.Positive Percent Agreement (PPA): 97.0% (385/397) (95% CI: 94.8% to 98.3%)Negative Percent Agreement (NPA): 90.6% (281/310) (95% CI: 86.9% to 93.4%)
Agreement with Predicate (Patients Considering Natalizumab)High Positive Percent Agreement and Negative Percent Agreement with the validated laboratory methodology used in clinical studies.Positive Percent Agreement (PPA): 98.5% (326/331) (95% CI: 96.5% to 99.4%)Negative Percent Agreement (NPA): 91.8% (268/292) (95% CI: 88.1% to 94.4%)
PML Risk Stratification (Clinical Utility)The assay should be statistically informative, meaning the percentage of positive results in patients with PML (pre-PML) is significantly higher than in the general MS population.The 100% JCV antibody positivity in 31 natalizumab-treated PML patients (pre-PML) was significantly different from the 58.7% JCV antibody positivity in the MS population, representing an approximately 2-fold increased risk of PML for JCV antibody positive individuals compared to the overall natalizumab-treated population. The relative risk of PML for JCV positive patients treated with ≥18 months of natalizumab was 30.4 (95% CI: 5.3 to 437.4) compared to JCV negative patients.
Reproducibility (Qualitative Results)Consistency in qualitative results (Negative, Indeterminate, Positive) across sites, days, runs/operators, and within the assay.High qualitative agreement (e.g., Negative Control: 89/90 ND, Positive Control: 90/90 D, Indeterminate Control: 90/90 I). Individual sample types (Plasma Low Positive, Serum Indeterminate) also showed high agreement, with varying levels of qualitative outcomes depending on the sample type's proximity to the cut-off.
Reproducibility (Quantitative Results)Low coefficient of variation (%CV) for quantitative results (OD and Index values) across sites, days, runs/operators, and within the assay.Overall %CV for Index values generally below 17%, with many values significantly lower (e.g., Positive Control Index: 3.7% total %CV; Indeterminate Control Index: 8.3% total %CV). For %Inhibition, total %CV generally below 12%.
Reproducibility at Lower Cut Point (%CV)Low %CV near the lower cut-point to demonstrate precision.Plasma: 2.3% %CV for Detection Assay (Index) and 2.2% %CV for Confirmation Assay (%Inhibition) near the lower cut-point.Serum: 2.6% %CV for Detection Assay (Index) and 2.0% %CV for Confirmation Assay (%Inhibition) near the lower cut-point.
Cross-Reactivity (Specific Antibodies)No detected reactivity with common antibodies (e.g., E. coli, M. tuberculosis, P. jiroveci).0/3 detected for all three antibodies tested (Antibody to Escherichia coli, Antibody to Mycobacterium tuberculosis, Antibody to Pneumocystis jiroveci).
Cross-Reactivity (Polyoma Viruses)No significant change in OD signal when spiked with BKV VLP, indicating no cross-reactivity with BKV.No samples exhibited >45% change in OD signal when spiked with BKV VLP (ranged from -15% to 27%), indicating no cross-reactivity with BKV.
Interference (Endogenous Substances)Observed differences in signal should not cause changes in interpretation for most common interferents (%Change from Baseline < 20%).Most interferents showed < 20% change from baseline (Albumin, Ascorbic Acid, Bilirubin, Cholesterol, Hemoglobin, Triglycerides).Gamma Globulin showed significant interference (831.6% in plasma, 758.5% in serum), which is expected due to high JCV antibody seroprevalence in normal human serum used for its production.
Hook EffectNo Hook Effect should be observed.No Hook Effect observed in the tested panel of serum samples ranging from negative to strong positive reactivity.
Sample Matrix Comparison (Sera vs. Plasma)High Positive and Negative Percent Agreement between serum and plasma, and a slope of 1 in Passing-Bablok regression.Positive Percent Agreement = 96.7% (58/60), 95% CI: (88.6 to 99.1%)Negative Percent Agreement = 97.9% (47/48), 95% CI: (89.1 to 99.6%)Passing-Bablok regression: slope of 1 (95% CI: 0.9928 to 1.0167) and intercept of 0 (95% CI: -0.0068 to 0.0016).
Sample Comparison (Fresh vs. Frozen)High Positive and Negative Percent Agreement between fresh and frozen samples, and a slope of 1 in Passing-Bablok regression.Positive Percent Agreement = 96% (72/75), 95% CI: (88.9 to 98.6%)Negative Percent Agreement = 96.7% (29/30), 95% CI: (83.3 to 99.4%)Passing-Bablok regression: slope of 1 (95% CI: 0.9900 to 1.0221) and intercept of 0 (95% CI: -0.0090 to 0.0028).

2. Sample Size Used for the Test Set and Data Provenance

  • PML Risk Stratification / Pre-PML samples:
    • Sample Size: 31 available serum samples from confirmed PML patients.
    • Data Provenance: Data collected from both clinical trial and post-marketing reports of confirmed cases of PML (retrospective, likely international given the nature of clinical trials for natalizumab, but not explicitly stated). Samples were collected at least 6 months prior to clinical diagnosis of PML.
  • Archived Clinical Specimens (Comparison to Predicate):
    • Sample Size:
      • Patients Receiving Natalizumab: 707 samples (resulted in 414 positive, 293 negative, for a total of 707 evaluable samples based on the table).
      • Patients Considering Natalizumab: 623 samples (resulted in 350 positive, 273 negative, for a total of 623 evaluable samples based on the table).
    • Data Provenance: Prospectively collected and archived clinical samples obtained from the STRATIFY-2 and AFFIRM clinical studies. The AFFIRM study included patients from "North America and EU/Rest of World," while STRATIFY-2 was conducted in the "US." This indicates a multi-national, prospective collection.
  • Reproducibility:
    • Sample Size: A panel consisting of four levels of sera and four levels of plasma (EDTA) samples, plus low and high positive controls. Each panel member was tested in three replicates, two runs a day for five days. The total number of quantitative results for Controls and plasma/serum samples listed in Table 5 is typically 90 or 120 per specific parameter, representing replicates across conditions.
    • Data Provenance: Not explicitly stated, but it describes a laboratory study, likely an internal development/validation study by Focus Diagnostics.
  • Reproducibility at the Lower Cut Point:
    • Sample Size: One contrived serum sample and one contrived plasma (EDTA) sample. Each was diluted 20 times and tested. (N=20 for plasma, N=19 for serum in the table, likely referring to the number of replicates after dilution).
    • Data Provenance: Laboratory study, likely internal.
  • Cross-Reactivity (Part One - Spiked Antibodies):
    • Sample Size: 3 replicates for each of 3 antibodies (total of 9 tests).
    • Data Provenance: Laboratory study.
  • Cross-Reactivity (Part Two - Sero-prevalence Comparison):
    • Sample Size: Remnant specimens for 13 different potential cross-reactants, with sample sizes ranging from 17 to 48 (Total 334 samples).
    • Data Provenance: Not explicitly stated, but these are "remnant specimens that previously tested positive for each potential cross reacting antibody," implying clinical samples.
  • Cross-Reactivity (Part Three - Polyoma Viruses):
    • Sample Size: 40 clinical specimens previously tested positive for JCV antibodies.
    • Data Provenance: Clinical samples.
  • Interferences:
    • Sample Size: Sera and plasma samples containing JCV antibody near the cut-off. Each interferent was tested (N not explicitly stated for each, but inferred from the "Average Index" values).
    • Data Provenance: Laboratory study.
  • Hook Effect:
    • Sample Size: A panel of serum samples "ranging from negative reactivity to strong positive reactivity" (not a specific number given).
    • Data Provenance: Laboratory study.
  • Sample Matrix Comparison:
    • Sample Size: 109 paired sera and plasma samples.
    • Data Provenance: Not explicitly stated, but likely clinical samples.
  • Sample Comparison - Fresh vs. Frozen:
    • Sample Size: 53 pairs of fresh and frozen plasma specimens and 53 pairs of fresh and frozen sera specimens (total 106 pairs).
    • Data Provenance: Not explicitly stated, but likely clinical samples.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not mention the use of experts to establish the ground truth for the test set.

  • For the comparative agreement studies, the "ground truth" for the test set (pre-PML samples and archived clinical specimens) was established by comparison to a "validated laboratory methodology" (the predicate device, STRATIFY JCV™ Antibody ELISA).
  • For PML risk stratification, clinical diagnosis of PML based on existing medical criteria was used as the outcome for the "pre-PML" samples.
  • The "control" samples for reproducibility and cross-reactivity studies were internally prepared or sourced as part of the laboratory validation process.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method involving multiple readers for the test set. The comparison studies rely on the results generated by the predicate device as the reference, which presumably has its own established interpretation and quality control.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size of Human Improvement with AI vs. Without AI Assistance

No MRMC comparative effectiveness study was done. This device is an in vitro diagnostic (IVD) assay (ELISA-based), not an AI-assisted diagnostic imaging or interpretation tool. Therefore, the concept of "human readers improve with AI vs. without AI assistance" does not apply to this specific device.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

The device is an immunoassay (ELISA) intended for "professional use only". It produces quantitative results (OD, Index values, %Inhibition) that are then interpreted against established cut-offs to yield qualitative results (Positive, Negative, Indeterminate). While the interpretation logic (cut-offs) is algorithmic, the test requires human involvement for sample preparation, running the assay, and reading the results from a spectrophotometer. The final interpretation of the test result (especially for indeterminate results requiring confirmation) and its use in risk stratification in conjunction with other clinical data explicitly involves a human healthcare professional. Therefore, it's not a standalone "algorithm only" device in the sense of a fully automated AI system rendering a diagnosis.

7. The Type of Ground Truth Used

The ground truth used varies based on the type of study:

  • Clinical Performance (PML Risk Stratification):
    • For pre-PML samples: Clinical diagnosis of PML (confirmed cases) combined with the patient's JCV antibody status by the predicate device.
    • For risk estimation: Outcomes data (incidence of PML) from natalizumab clinical trials and post-marketing reports, stratified by JCV serological status.
  • Comparative Agreement Studies (Archived Clinical Specimens):
    • Results from the predicate device (STRATIFY JCV™ Antibody ELISA), which is a validated laboratory methodology.
  • Reproducibility, Cross-Reactivity, Interferences, Hook Effect, Sample Matrix/Fresh vs. Frozen:
    • Reference materials, contrived samples, or previously characterized clinical samples with known (or expected) JCV antibody status or presence of interfering/cross-reacting substances. These are essentially "expert consensus" or "reference method" results for analytical performance.

8. The Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of machine learning. As this is an ELISA assay, the development and optimization process would involve analytical studies to determine optimal reagent concentrations, incubation times, and optical density (OD) readings. The cut-off values (Index < 0.2, negative; Index ≥ 0.4, positive; 0.2-0.4 indeterminate) and Confirmation Assay cut-offs are likely established through extensive analytical validation and clinical correlation studies, but these are not explicitly termed "training set" data.

9. How the Ground Truth for the Training Set Was Established

As noted above, there is no explicit "training set" described as such. The establishment of cut-off values and assay parameters would typically be done through:

  • Extensive testing of characterized clinical samples: This involves samples with known JCV antibody status (determined by a gold standard or comprehensive clinical assessment), alongside samples representing different levels of antibody reactivity.
  • Statistical analysis: To define thresholds that optimize sensitivity and specificity.
  • Correlation with clinical outcomes: As demonstrated by the PML risk stratification data, where the assay's results correlated with the incidence of PML, thus validating the clinical relevance of the chosen cut-offs.

The predicate device and its established performance also served as a critical benchmark in defining the desired performance characteristics during the development of STRATIFY JCV® DxSelect™.

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STRATIFY JCV @ DxSelect™ (EL1950) Page 1 of 10

ApplicantFocus Diagnostics, Inc.11331 Valley View StreetCypress, California 90630USAAUG 16 2012
Establishment Registration No.2023365
Contact PersonTara Vivianitel 562-240-6115fax 562.240.6530tviviani@focusdx.com
Summary DateJuly 24, 2012
Proprietary NameSTRATIFY JCV ® DxSelect™
Generic NameJCV ELISA
ClassificationClass II, Special Controls§ 21 CFR 866.3336, Anti-JCV antibody detection assay.Product Code: OYP
Predicate DevicesSTRATIFY JCV™ Antibody ELISA

INTENDED USE

The Focus Diagnostics' STRATIFY JCV® DxSelect™ assay is intended for the qualitative detection of antibodies to JC virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis patients receiving or considering natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only.

The assay is not intended for donor screening. The performance of this assay has not been established for use in other immunocompromised patient populations or patients with different disease conditions or undergoing other treatments or in neonates and pediatric patient populations.

SUMMARY AND EXPLANATION OF THE TEST

Progressive multifocal leukoencephalopathy (PML) is an opportunistic infection of the central nervous system (CNS) caused by JC virus (JCV), a polyoma virus, that is pathogenic only in humans. It is thought that exposure to the JC virus occurs early in life (pre-adolescence) and recently published studies have reported that approximately 50% to 60% of adults have been infected with JCV, as evidenced by the presence of antibody to JCV in the serum. In rare instances, the virus reactivates and progresses to PML such as in individuals who are immune compromised. Treatment with immunomodulatory therapies increase the risk of developing PML in JCV infected individuals. While JCV exposure is necessary for the development of PML, the development of the disease is also dependent on both host and viral factors, as well as immune status. Since JCV infection is a necessary step for PML development, an assay to detect JCV exposure in patients may be a potentially useful tool to stratify patients for PML risk (i.e., identifying patients who may be at a lower or higher risk of developing PML).

There is an increased risk of PML in natalizumab treated multiple sclerosis (MS) and Crohn's disease (CD) patients. Three risk factors for PML have been identified in MS and CD patient populations: natalizumab treatment duration, prior immunosuppressant use, and the presence of antibodies to JCV. Utilizing these three risk factors, sub-groups of patients can be identified with both higher and lower risk for PML. Please consult the current, locally available, prescribing or supplemental physician information for natalizumab (Tysabri®) for detailed information on the known risks associated with JCV serological status and the development of PML in natalizumab-treated patient's JCV antibody status should be considered in combination with other known PML risk factors when evaluating the benefit and risk of initiating or continuing therapy with natalizumab. Patients with all three known risk factors have the highest risk for the development of PML.

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120986 510(k) Summarv STRATIFY JCVg DxSelect™ (EL1950) Page 2 of 10

TEST PRINCIPLE

In the Focus Diagnostics' STRATIFY JCVg DxSelect™ test, JC virus-like particles (VLP) are pre-coated onto 96-well microiter plates. Diluted serum or plasma specimens and controls are incubated in the wells to allow JCV-specific antibodies present in the specimens to react with the JC VLP antigen. Nonspecific reactants are removed by washing. Peroxidase-conjugated anti-human antibodies are added to react with JCV-specific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of optical density (OD). Specimen OD readings are compared with Cut-Off Calibrator OD readings to determine results. Each specimen result is reported as an index value. A specimen with an index value that is greater than a specified upper cut-off is reported as positive for detectable JCV-specific antibodies, whereas a specimen with an index value less than the specified lower cut-off is reported as negative for detectable JCV-specific antibodies. A specimen with an index value that is equal to or between the upper and lower cut-off values is reported as indeterminate. An indeterminate result requires further evaluation in the confirmation (inhibition) assay.

In the confirmation assay, soluble JC VLP antigen will compete with plate bound JC VLP antigen for the JCV-specific antibodies present in the serum or plasma specimens. After washing away the unbound antibodies, peroxidase-conjugated anti-human antibodies are added and bind to any captured JCVspecific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromagen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of OD. The percent inhibition is calculated to confirm presence of JCV-specific antibodies in the specimens with a percent inhibition value that is greater than the specified cut-off are reported as positive for detectable JCV-specific antibodies, whereas specimens with percent inhibition values less than or equal to the cut-off are reported as negative for detectable JCV-specific antibodies.

ItemDevicePredicate
NameSTRATIFY JCV® DxSelect™STRATIFY JCV™ Antibody ELISA
Intended UseThe Focus Diagnostics' STRATIFYJCV® DxSelect™ assay is intended forthe qualitative detection of antibodies toJC virus in human serum or plasma.The assay is intended for use inconjunction with other clinical data, inmultiple sclerosis patients receiving orconsidering natalizumab therapy, as anaid in risk stratification for progressivemultifocal leukoencephalopathydevelopment. The assay is forprofessional use only.The assay is not intended for donorscreening. The performance of thisassay has not been established for usein other immunocompromised patientpopulations or patients with differentThe STRATIFY JCV™ Antibody ELISAtesting service provided by FocusDiagnostics is intended for thequalitative detection of antibodies toJohn Cunningham Virus in humanserum or plasma. The assay isintended for use in conjunction withother clinical data, in multiple sclerosisand Crohn's disease patients receivingnatalizumab therapy, as an aid in riskstratification for progressive multifocalleukoencephalopathy development.The assay is for professional use onlyand is to be performed only at FocusDiagnostics' Reference Laboratory.The assay is not intended for donorscreening. The performance of thisassay has not been established for use
disease conditions or undergoing othertreatments or in neonates and pediatricpatient populations.in other immunocompromised patientpopulations or in neonates andpediatrics patient populations.
Assay MethodologyDetection ELISA with a secondaryConfirmation ELISA for specimens thathave results between the two cut-offlevels.Detection ELISA with a secondaryConfirmation ELISA for specimens thathave results between the two cut-offlevels.
Assay TargetAntibodies to John Cunningham Virus(JCV)Antibodies to John Cunningham Virus(JCV)
Sample Type(s)Serum and plasmaSerum and plasma

Comparison to Predicate

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K120986

STRATIFY JCVg DxSelect™ (EL1950) Page 3 of 10

ItemDevicePredicate
NameSTRATIFY JCV® DxSelect™STRATIFY JCV™ Antibody ELISA
Sample Preparation1:101 dilution in sample diluent1:200 dilution in sample diluent
Detection Assay- Confirmation Assay1:101 dilution in confirmation diluent1:200 dilution in assay diluent spiked with JCV antigen.
Assay Cut-OffsIndex < 0.2, negativeIndex ≥ 0.4, positivenOD < 0.1, negativenOD ≥ 0.2, positive
Assay PreparationELISA plates are pre-coated, all buffers are pre-mixed. Calibrators and controls require dilution before use.User must coat ELISA plates prior to use and mix specific reagents.Controls require dilution before use.
AntigenJC Virus Like ParticlesJC Virus Like Particles
AntibodyPeroxidase-conjugated donkey anti-human JCV bodiesPeroxidase-conjugated donkey anti-human JCV bodies

Clinical Performance (Comparative Agreement)

Because PML is an infrequent event in natalizumab-treated patients, data collected from both clinical trial and post-marketing reports of confirmed cases of PML were used to assess the clinical performance of the STRATIFY JCVg DxSelect™ assay for PML risk stratification. A clinical plan was developed for collection of serum samples obtained from natalizumab-treated patients prior to the onset of PML for JCV antibody testing. A total of 31 available serum samples from confirmed PML patients collected at least 6 months prior to clinical diagnosis of PML were tested for JCV antibody status using the STRATIFY JCV6 DxSelect™ assay. In addition to the pre-PML samples, 1330 Samples from MS patients were tested by the STRATIFY JCV DxSelect™ assay. A total of 707 patients receiving treatment tested positive for antibodies to JCV using the test, and the positivity rate was estimated to be 58.7% (414/707) with a 95% CI of 54.9% to 62.1%. An assay is statistically informative if the percentage of positive results in patients with the disease of interest is higher than the percentage of positive results in the population at risk. The 100% JCV antibody positivity demonstrated in the 31 natalizumab-treated PML patients prior to PML diagnosis was significantly different than the 58.7% JCV antibody positivity in the MS population, and represents an approximately 2-fold increased risk of PML compared to the PML incidence in the overall natalizumab-treated population.

In prior clinical studies for natalizumab the risk of developing PML was estimated using statistical modeling. The relative risk for patients who have received natalizumab for at least 18 months is shown in the table below. Risk was calculated based on statistical modeling with an assumption that there is one hypothetical PML case with a negative test result (38 PML cases: 37 positive and 1 hypothetical negative) and an assumption that the study has 13,227 patients.

Number withPMLNumber withoutPMLTotal numberpatients treated
JCV Serological StatusPositive377,2297,266
Negative1*5,9605,961
Total3813,18913,227
Risk of PML (per 1,000) treated with ≥ 18months for Positive result5.0995% CI: 3.70 to 7.01
Risk of PML (per 1,000) treated with ≥ 18months for Negative result0.1795% CI: 0.03 to 0.95
Relative risk30.495% CI: 5.3 to 437.4

Table 1: Estimated incidence of PML by JCV serological status

"For the negative result, a hypothetical case was assumed in order to allow for calculation of the point estimate.

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510(k) Summarv STRATIFY JCVg DxSelect™ (EL1950) Page 4 of 10

12098

The studies demonstrated that the positivity rate for JCV antibodies is not dependant upon prior immunosuppressant (IS) use or the duration of natalizumab treatment. The results of the STRATIFY JCV @ DxSelect™ assay can be used along with other established PML risk factors, of prior IS use and natalizumab treatment duration, to stratify an individuals risk for PML, please refer to Table 1 and to the prescribing information for additional risk estimates.

Performance with pre-PML samples.

The STRATIFY JCVg DxSelect™ assay was compared to a cleared Anti-JCV assay (STRATIFY JCV™ Antibody ELISA) using serum samples obtained from PML patients at least 6 months prior to PML diagnosis. Because PML is an infrequent event in natalizumab-treated patients, data collected from both clinical trial and post-marketing reports of confirmed cases of PML were used to assess the clinical performance of the STRATIFY JCVg DxSelect™ assay for PML risk stratification. Thirty-one available serum samples from confirmed PML patients collected at least 6 months prior to clinical diagnosis of PML were tested at one internal testing site for JCV antibody status using the STRATIFY JCV。DxSelect™ assay and the validated laboratory methodology. The sample set demonstrated 100% (31/31) positive agreement (95% CI: 89.0% to 100%) with the validated assay.

Performance with archived clinical specimens

Two groups of prospectively collected and archived clinical samples obtained from the STRATIFY-2 and the AFFIRM clinical studies were used to assess the performance of the STRATIFY JCVg DxSelect™ assay compared to the validated laboratory methodology used in the STRATIFY-2 and AFFIRM clinical studies. One group consisted of patients who were receiving natalizumab, the other group of patients had not received natalizumab therapy (and were considering natalizumab). The samples were blinded and randomly distributed to two external testing sites and one internal testing site. The data was analyzed for each group separately. Results for the two individual groups are presented in the tables Positive Percent Agreement was greater than 97% for each group and Negative Percent below. agreement was greater than 90% for each group.

STRATIFY JCV® DxSelect™Cleared Anti-JCV AssayTotal
PositiveNegative
Positive38529414
Negative12281293
Total397310707
Positive Percent Agreement (PPA)97.0% (385/397)95% CI: 94.8 to 98.3%
Negative Percent Agreement (NPA)90.6% (281/310)95% CI: 86.9 to 93.4%

Table 2: Agreement for Clinical Samples - Patients Receiving Natalizumab

Table 3: Agreement for Clinical Samples - Patients Considering Natalizumab

STRATIFY JCV® DxSelect™Cleared Anti-JCV AssayTotal
PositiveNegative
Positive32624350
Negative5268273
Total331292623
Positive %Agreement (PPA)98.5% (326/331)95% CI: 96.5 to 99.4%
Negative %Agreement (NPA)91.8% (268/292)95% CI: 88.1 to 94.4%

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Image /page/4/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of a stylized, curved shape on the left, resembling a swoosh or a crescent. To the right of this shape is the word "FOCUS" in bold, sans-serif capital letters. Below "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined.

STRATIFY JCV% DxSelect Page 5 of 10

Positivity Rate and Expected Values:

The STRATIFY JCV% DxSelect™ assay been used to evaluate JCV antibody positivity rate in serum and plasma samples from a geographically diverse cohort of 1330 MS patients. The cohort was comprised from MS patients from clinical trials including a completed Phase 3 clinical study of natalizumab in MS patients (AFFIRM C-1801), an ongoing study to evaluate seroprevalence in the MS population (STRATIFY-2 [101JC402). The clinical characteristics for the MS patients within each study are shown in Table 4. The age and gender distribution of the MS cohort tested with the STRATIFY JCV% DxSelect™ are similar to the age and gender distribution of MS patients treated with natalizumab in the postmarketing setting. JCV antibody positivity rate in the MS cohort was 55-59% which is consistent with what has been reported in the literature. JCV antibody positivity rate was shown to increase with age and was lower in women compared to men which is also consistent with what has been reported in the literature in healthy adults using similar assay methodologies.

Seroprevalence data was evaluated for each study. The observed seroprevalence using the STRATIFY JCVa DxSelect™ assay was 59% (467/792) in the STRATIFY-2 study and 55% (296/538) in the AFFIRM Study. These seroprevalence values are consistent with the seroprevalence values observed during the clinical studies, when the relationship between JCV serological status and PML development were described. Additionally the STRATIFY JCVg DxSelect™ assay demonstrated 100% concordance with 31 pre-PML samples.

AFFIRM(N=538)STRATIFY-2(N=792)
Age (years)
• Range18-5019-78
• Mean35.846.4
• Median3646
Gender (%)*
• Male173/538 (32.2%)202/792 (25.5%)
• Female365/538 (67.8%)590/792 (74.5%)
GeographyNorth America and EU/Rest ofWorldUS
JCV Antibody Positivity Rate(95% CI)
• % JCV Antibody Positive297/538 (55.2%)(51.0 to 59.4)467/792 (59.0%)(55.5 to 62.3)
• % JCV Antibody Negative241/538 (44.8%)(40.6 to 49.0)325/792 (41.0%)(37.7 to 44.5)

Table 4: Demographic Data and JCV Antibody Prevalence for MS Patients

  1. A total of 10.4% of the samples tested in the AFFIRM study and 16.4% of the samples tested in the STRATIFY-2 study were indeterminate in the DETECTION ASSAY.

Reproducibility

The reproducibility of the assay was assessed using a protocol based on a CLSI guideline for estimating precision of an assay. The protocol consisted of testing three replicates of each panel member for two runs a day for a total of five days at two external testing sites an one internal site. The reproducibility testing panel consisted of four levels of sera and four levels of plasma (EDTA) samples and the low and high positive controls. The four levels included a negative, low, indeterminate and moderate positive sample prepared in a serum matrix and a negative, low, indeterminate and moderate positive sample prepared in a plasma matrix. The indeterminate samples were tested simultaneously in the Confirmation Assay and the Detection Assay.

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Image /page/5/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of the word "FOCUS" in bold, sans-serif font, with a curved, swooping line above it. Below the word "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined with a thin line.

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510(k) Summarv STRATIFY JCVg DxSelect™ (EL1950) Page 6 of 10

Table 5: Reproducibility

ParameterSampleMatrixSampleNameQualitativeResultsQuantitative Results
NDIDNVTotalNMeanBetweenSitesBetweenDaysBetweenRuns/OperatorsWithin Assay(Repeatability)Total
SD%CVSD%CVSD%CVSD%CVSD%CV
ODCut-Off1201.1310.14512.80.0675.90.0887.80.0373.20.18616.5
INDEXControlsNegativeControl89190890.1020.0109.50.0077.10.0109.70.0066.10.01716.5
PositiveControl9090901.2020.0080.70.0100.80.0221.80.0363.00.0443.7
Indeterminate9090900.2700.0103.70.0093.40.0114.20.0145.10.0238.3
PlasmaLow positive395190900.4100.0000.00.0000.00.0225.50.0235.70.0327.9
Mediumpositive9090900.8580.0414.70.0253.00.0182.10.0596.90.078
SerumNegative89190890.1290.0021.80.0075.50.0139.80.0085.80.01712.8
Indeterminate9090900.2830.0124.30.0000.00.0113.90.0186.30.0248.5
%InhibitionPlasmaLow positive187290900.4220.0040.90.0041.10.0184.20.0133.10.0235.4
Mediumpositive9090901.0550.0484.50.0000.00.0413.90.0454.30.078
SerumNegative842490860.1090.02422.30.0000.00.01816.50.01311.70.03330.1
Indeterminate11772908851.052.0063.90.0000.03.6487.13.3576.65.34810.5
SerumIndeterminate2853908762.593.8896.22.4383.90.0000.05.5508.97.20211.5

ND = Not Detected, I = Indeterminate, D = Detected, NV = Invalid

Reproducibility at the Lower Cut Point

In order to demonstrate precision near the lower cut point, two contrived samples, one sera and one plasma (EDTA) were prepared to be near the lower cut-point of the assay. Each sample was diluted twenty times and tested at one internal site in the Detection Assay and the Confirmation Assay. As depicted in the following table the %CV was ≤ 2.6%.

Table 6: Reproducibility at the Lower Cut Point
--------------------------------------------------------
SampleMatrixDescriptive Statistics of Detection Assay(Index)Descriptive Statistics of Confirmation Assay(%Inhibition)
NMinMaxMeanSD%CVNMinMaxMeanSD%CV
Plasma200.330.360.340.012.32061.6666.9765.111.432.2
Serum19*0.190.210.20.012.61955.8960.92581.162

*One replicate was Invalid

.

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Image /page/6/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo features a stylized, curved shape resembling an eye or a crescent moon on the left side. To the right of this shape is the word "FOCUS" in bold, sans-serif font. Below "FOCUS" is a horizontal line, and beneath that line is the word "Diagnostics" in a smaller, sans-serif font.

Cross Reactivity

Cross reactivity was evaluated in a three part study conducted at an internal testing site. Part one of the study evaluated cross reactivity with commercially available human antibodies spiked into JCV negative serum and plasma as determined by the STRATIFY JCV DxSelect™ assay. The potentially cross reacting antibodies were spiked in at concentrations estimated to be approximately 2-4 times higher than the estimated limit of detection of JCV antibody. Each antibody tested was not detected with the STRATIFY JCV DxSelect™ assay. There was no observed reactivity with the three potentially crossreacting antibodies tested in part one of the study.

Table 7: Cross Reactivity - Part One - Spiked Antibodies

Cross ReactantConcentrationNumber DetectedSerumNumber DetectedPlasma
Antibody to Escherichia coli0.4 µg/mL0/30/3
Antibody to Mycobacterium tuberculosis0.4 µg/mL0/30/3
Antibody to Pneumocystis jiroveciunquantified0/30/3

In part two of the study panel consisted of a least twenty remnant specimens that previously tested positive for each potential cross reacting antibody. Each member of the panel was evaluated using the STRATIFY JCVം DxSelect™ assay along with appropriate controls. The seroprevalence of JCV for each group of potential cross reactants in the panel was compared to the expected seroprevalence of JCV in the normal population. If a group demonstrated a higher than expected seroprevalence of JCV (>70%) it may be an indicator of potential cross reactivity. Four groups of patients (C. pneumoniae, HIV, CMV, HSV 1) exhibited a positivity rate that was slightly above that observed in previously reported studies. The results suggest that these groups demonstrate potential cross reactivity.

SampleMatrixCross ReactantTotal No.ofReplicatesorSamplesScreeningResult Count(Based onIndex)ConfirmationResult Count(Based on%Inhibition)Final Interpretation%
DINDNDNotTestedDNDDNDDND
UnknownHIV221624201117577.322.7
C. pneumoniae4835584323371177.122.9
SerumC. trachomatis201127181112860.040.0
CMV402776335232880.020.0
Candida4026683415271367.532.5
EBV4023983127251562.537.5
HSV 148326104224341470.829.2
HSV 220857151491145.055.0
HHV 620112718211955.045.0
Listeria178361438947.152.9
Mycoplasma201046164101050.050.0
Treponema pallidum191225171113668.431.6
VZV201244161313765.035.0
All3342084878286153322311166.833.2

Table 8: Cross Reactivity - Part Two -- Sero-prevalence Comparison

D= Detected, ND = Not Detected, IND = Indeterminate

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STRATIFY JCVg DxSelect™ (EL1950) Page 8 of 10

Part three of the cross reactivity evaluation consisted of an evaluation of the potential cross reactivity with other polyoma viruses. This included a cross absorption study using BKV virus like particles (VLP). A total of 40 clinical specimens that have previously tested positive for JCV antibodies in the STRATIFY JCV% DxSelect™ assay were tested in a confirmation style assay using JCV VLP and BKV VLP. A control set consisted of samples spiked with JCV VLP at the same concentration of VLP that is used in the confirmation assay. The test samples were with BKV VLP at the same concentration. The tests samples would be considered to be cross reactive if the % change in signal between the unspiked sample and the sample spiked with BKV VLP is > 45%. The control set demonstrated % change in OD that was consistent with expectations. The test samples demonstrated % change in OD values due to BKV VLP that ranged from -15 to 27%. No samples exhibited >45% change in the OD signal when spiked with BKV VLP, indicating that the assay does not cross react with BKV.

Additional analysis of the structure of the VP1 protein of other polyoma viruses indicates that BKV and JCV are more closely related (79.4% similarity) than other polyoma virus such as Merkel Cell virus and WU virus and KI virus (30.8 to 50.8 % similarity). Due to the differences in viral structure a similar experiment with other polyoma virus VLP was not conducted.

Interferences

Potential interference due to endogenous substances were evaluated using a protocol based on a CLSI guideline for estimating the effect of interfering substances. The testing panel consisted of sera and plasma samples that contain JCV antibody with an index value that is close to the assay cut off. The samples are spiked with the potential interferent at the highest possible endogenous level and compared to baseline testing of the same serum and plasma samples that did not contain the interferent. For all of the potential interferents with the exception of y globulin; the observed differences in signal did not cause any changes in interpretation of the final result. A potentially interference is suspected if the % Change from the baseline sample is > 20%. Commercially available y globulin is produced using normal human serum containing IgG antibodies, since the seroprevalence of antibodies to JCV virus is approximately 55% in the normal population it is expected to react with this assay.

SubstanceNameSubstanceConcentrationPlasmaSerum
Average IndexBaselineSampleAverage IndexInterferenceSample%ChangefromBaselineAverage IndexBaselineSampleAverage IndexInterferenceSample%ChangefromBaseline
Albumin120 mg/mL0.350.388.60.430.37-14.0
Ascorbic Acid0.03 mg/mL0.400.425.00.420.444.8
Bilirubin0.2 mg/mL0.330.330.00.420.38-9.5
Cholesterol5 mg/mL0.480.40-16.70.460.44-4.3
Gamma Globulin60 mg/mL0.383.54831.60.413.52758.5
Hemoglobin110 mg/mL0.4113.50.465.0
Hemoglobin165 mg/mL0.360.386.10.440.39-10.1
Hemoglobin220 mg/mL0.35-2.40.37-16.0
Triglycerides10 mg/mL0.360.35-2.80.400.37-7.5

Table 9: Interference Summary - Signal Comparison to Baseline

Note: Three dilution of Gamma Globulin stock solution exhibited Index values - 3.22, 3.57 and 3.40.

HOOK EFFECT

The Hook Effect was investigated during the optimization of the JCV VLP coating concentration and conjugate dilution using a panel of serum samples ranging from negative reactivity to strong positive reactivity (beyond the limit of the plate reader at OD450 - 4.000) to JCV. No Hook Effect was observed at the optimal coating VLP concentration and conjugate dilution for this assay using the panel tested.

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510(k) Summarv STRATIFY JCV% DxSelect™ (EL1950) Page 9 of 10

<120986

SAMPLE MATRIX COMPARISON

A panel of 109 paired sera and plasma samples (EDTA) were evaluated in the STRATIFY JCV6 DxSelect™ assay. Of the total 109 pairs one pair was eliminated from the regression analysis because the result of the sera sample was invalid, of the remaining 108 pairs 7 pairs were qualitatively detected with OD values that exceeded the range of the spectrophotometer. These pairs of samples are included in the qualitative result comparison below, but eliminated from the regression analysis since an index value could not be calculated. Passing-Bablok regression analysis of the pairs of sera and plasma specimens demonstrates a slope of 1 with a 95% Cl of (0.9928 to 1.0167)) and an intercept of 0 with a 95% Cl of (-0.0068 to 0.0016). Analysis of the qualitative results yielded Positive Percent Agreement = 96.7% (58/60), 95% Cl: (88.6 to 99.1%), and Negative Percent Agreement = 97.9% (47/48), 95%Cl: (89.1 to 99.6%)

Final Plasma ResultFinal Serum Result (Count)All
NDDInvalid
ND472150
D15859
All48601109

Figure 1: Passing-Bablok Regression Analysis (Sera vs. Plasma)

Sample Comparison -Fresh vs. Frozen

A panel of 53 pairs of fresh and frozen plasma specimens (EDTA) and 53 pairs of fresh and frozen sera specimens were evaluated in the STRATIFY JVC DxSelect assay. Of the total 106 pairs, two serum pairs and three plasma pairs were excluded from the regression analysis due to having OD readings above the spectrophotometers measuring range. One pair of serum samples was excluded from the regression analysis due to an invalid result for the fresh specimen. Passing-Bablok regression analysis of the pairs of fresh vs. frozen specimens demonstrates a slope of 1 with a 95% Cl of (0.9900 to 1.0221) and an intercept of 0 with a 95% Cl of (-0.0090 to 0.0028). Analysis of the qualitative results vielded a Positive

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Percent Agreement = 96% (72/75), 95% C1: (88.9 to 98.6%) and Negative Percent = 96.7% (29/30), 95%CI: (83.3 to 99.4%).

Final Frozen Result (Count)
Sample MatrixFinal Fresh ResultNDDAll
PlasmaND1616
D13637
All173653
SerumND13114
D23638
NV11
All1616
All3268106

Table 11: Sample Comparison -Fresh vs. Frozen

ND = Not Detected, D = Detected; NV = Invalid

  • Three plasma pairs and two serum pairs had results that were above the range of the spectrophotometer, these samples are considered detected.

Figure 2: Sample Comparison -Regression Analysis Fresh vs. Frozen

Image /page/9/Figure/8 description: The image shows a Passing-Bablok Regression Fit with Confidence Bound. The regression line is defined as P-B Reg Line = 0 + 1 * X. The 95% confidence interval for the intercept is (-0.0090,0.0028), and the 95% confidence interval for the slope is (0.9900,1.0221). The sample size is n=100, and the data points are separated into serum and plasma.

Note: Two serum pairs and three plasma pairs were excluded from the regression analysis due to having OD readings above the spectrophotometers measuring range. One pair of serum samples was excluded from the regression analysis due to an invalid result for the fresh specimen.

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Image /page/10/Picture/1 description: The image is a circular seal for the Department of Health & Human Services in the USA. The seal features the department's name in a circular arrangement around the perimeter. In the center of the seal is a stylized emblem, possibly representing a bird or a symbolic figure, with flowing lines.

Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993

AUG 16 2012

Focus Diagnostics, Inc. C/O Tara Viviani 11331 Valley View Street Cypress, California 90630

Re: K120986

Trade/Device Name: STRATIFY JCV @ DxSelect™ Regulation Number: 21 CFR 866.3336 Regulation Name: John Cunningham Virus serological reagents Regulatory Class: Class II Special Controls Product Code: OYP Dated: August 3, 2012 Received: August 6, 2012

Dear Ms. Viviani:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. .

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice

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Page 2 - Ms. Tara Viviani

requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed nredicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known):

STRATIFY JCV® DxSelect™ Device Name:

Indications for Use:

The Focus Diagnostics' STRATIFY JCV® DxSelect™ assay is intended for the qualitative detection of antibodies to JC virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis patients receiving or considering natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only.

The assay is not intended for donor screening. The performance of this assay has not been established for use in other immunocompromised patient populations or patients with different .disease conditions of undergoing other treatments or in neonates and pediatric patient populations.

Prescription Use _ X (Part 21 CFR 801 Subpart D) And/Or

Over-The-Counter Use (21 CFR 807 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

. Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OVD)

Taura Feldblez
Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K120986

§ 866.3336 John Cunningham Virus serological reagents.

(a)
Identification. John Cunningham Virus serological reagents are devices that consist of antigens and antisera used in serological assays to identify antibodies to John Cunningham Virus in serum and plasma. The identification aids in the risk stratification for the development of progressive multifocal leukoencephalopathy in multiple sclerosis and Crohn's disease patients undergoing natalizumab therapy. These devices are for adjunctive use, in the context of other clinical risk factors for the development of progressive multifocal leukoencephalopathy.(b)
Classification. Class II (special controls). The special control for this device is the FDA guideline document entitled “Class II Special Controls Guideline: John Cunningham Virus Serological Reagents.” For availability of the guideline document, see § 866.1(e).