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BK110011 Summary

LIFECODES Class II IDv2, Enhanced
510(k) Summary

I. Submitter:

Owner's Name: Gen-Probe GTI Diagnostics, Inc.
Address: 20925 Crossroads Circle, Waukesha, WI 53186
Phone: 262.754.1000
Fax: 262.754.9831
Name of Contact Person: Ben Boldt, Ph.D.
Date Summary Prepared: June 04, 2012

II. Name of Device:

Device Name: LIFECODES Class II IDv2
Proprietary Name: LM2Q
Classification Name: Unclassified
Product Code: None

III. Name of Predicate Device for Claiming Equivalence

LIFECODES Class II ID (BK040035)

IV. Description of Device

Human Leukocyte antigens (HLA) are a system of glycoproteins that have a functional role in the presentation of peptides to the immune system. However, as a highly polymorphic system HLA molecules can become targets of antibody responses in people during pregnancy, transfusion of blood products or organ transplant rejection. Generally, alloimmunization leads to the production of HLA antibodies in approximately 33% of exposed individuals. The presence or absence of these HLA-specific antibodies has a role in determining the survival of transplant allografts.

LIFECODES Class II IDv2 is a bead-based immunoassay that uses the Luminex technology to qualitatively detect antibodies to Class II HLA. LIFECODES Class II IDv2 is designed to be used on either the Luminex100 or Luminex 200 platform. The Luminex instrument has been previously cleared by FDA (K073506).

LIFECODES Class II IDv2 is a modification of the previously FDA cleared LIFECODES Class II ID (BK 040035). Both products were designed and developed and are manufactured by Gen-Probe GTI Diagnostics for Gen-Probe TransplantDiagnostics.

The modifications included in LIFECODES Class II IDv2 were intended to improve the capability of the product to detect antibodies to Class II HLA, specifically to better detect antibodies specific to antigens encoded in the DQ locus of Class II HLA. To accomplish this, the design of the LIFECODES Class II IDv2 product was modified in the following ways:

The current LIFECODES HLA-specific antibody assay, LIFECODES Class II ID, uses 30 different unique bead populations conjugated with Class II HLA affinity purified from different cell lines. The HLA class II-conjugated beads are combined with 4 control beads to generate a bead blend.

LIFECODES Class II IDv2 contains the same class II HLA beads that are currently in LIFECODES Class II ID and adds 12 new beads to the bead blend. The additional HLA for each of the new beads is also affinity purified from cell lines and is enriched for antigen from the DQ locus.

Other than the addition of the new beads, the remaining kit components used in the LIFECODES Class II IDv2 assay are identical to the components of the LIFECODES Class II ID assay.

V. Principles of Operation

The principle of operation and the specific steps used in the LIFECODES Class II IDv2 assay are identical to those of the LIFECODES Class II ID assay. Both assays are performed manually using a microwell filter plate and the signal generated from the assay is measured using the Luminex instrument. Briefly, an aliquot of the bead blend (5μL) is incubated for 30 minutes at room temperature with the serum sample (12.5 μL) which has been diluted 1:4 in Wash Buffer. After the incubation, the beads are washed by aspiration to remove unbound antibody. An anti-human IgG antibody conjugated to phycoerythrin is then added. After incubation for 30 minutes at room temperature, the beads are aspirated and resuspended in 150 μL of Wash Buffer and fluorescence from each bead is measured using the Luminex instrument.

To determine if a sample is positive or negative a number of calculations are performed; the first of which is to determine if a specific HLA bead is positive or negative for bound alloantibody. To do this the signal intensity (Median Fluorescence Intensity or MFI) of the specific HLA conjugated bead is compared to the signal intensity of each of the three different control beads and the CalcCON (based on the the lowest antigen-conjugated bead MFI+ 20), generating 4 separate ratios of reactivity for each bead in the bead blend. A specific cutoff based on an adjusted value (background adjustment factor) is then applied to each of the four ratios for each HLA bead. Ratios that are greater than or equal to the cutoff are considered to be positive and ratios that are less than the cutoff are considered negative. An HLA bead is considered positive if two out of the four ratios are positive.

There are three quality control checks for the LIFECODES Class II IDv2 assay. The first two include the use of the Positive Sera Control and a Negative Sera Control provided in the kit. The Positive and Negative Control Sera are required to be tested in separate wells each time the assay is performed. In order for the results of the assay using the test sample to be considered valid, the Positive Control Serum must demonstrate a positive result and the Negative Control Serum must demonstrate a negative result. The requirements for the sera controls are intended to demonstrate that the user correctly performed the assay. The third control uses the results of the reaction with Probe 77 when testing with the kitsupplied control sera. Probe 77 which consists of Human IgG conjugated to a bead population must show positive reactivity of greater than 10,000 MFI in order for the assay results to be valid. This demonstrates that sufficient conjugate was added to the assay.

VI. Intended Use

LIFECODES Class II IDv2 is a bead-based immunoassay used to qualitatively detect Panel Reactive HLA IgG antibodies (PRA). This kit is intended for In Vitro Diagnostic (IVD) use.

VII. Support of substantial equivalence based on comparison of features, characteristics and components to the predicate device:

PRODUCT
CHARACTERISTIC
LifeCodes ID Class IILifeCodes ClassII IDv2
Intended UseLifeCodes ID Class II is a bead-based immunoassay used to qualitatively detect Panel Reactive HLA IgG antibodies (PRA). This kit is intended for In Vitro Diagnostic (IVD) use.LifeCodes Class II IDv2 is a bead-based immunoassay used to qualitatively detect Panel Reactive HLA IgG antibodies (PRA). This kit is intended for In Vitro Diagnostic (IVD) use.
Indications for UseLifeCodes ID Class II is a qualitative immunoassay that can be used to detect the presence of IgG antibodies produced in patients sensitized to HLA by transfusion, pregnancy, or tissue transplantation. This kit is intended for In Vitro Diagnostic (IVD) use.LifeCodes Class II IDv2 is a qualitative immunoassay that can be used to detect the presence of IgG antibodies produced in patients sensitized to HLA by transfusion, pregnancy, or tissue transplantation. This kit is intended for In Vitro Diagnostic (IVD) use.
TechnologyBead based immunoassay using the Luminex technologyBead based immunoassay using the Luminex technology
Reportable ResultsQualitative assay; results are reported as positive or negativeQualitative assay; results are reported as positive or negative
Sample TypeSerumSerum

 

KIT COMPONENTSLifeCodes ID Class IILifeCodes ClassII IDv2
Bead Blend – HLAConjugated Beads30 beads conjugated with affinity purified class II antigen derived from donors

30 beads conjugated with affinity purified class II antigen derived from donors

12 additional beads conjugated with purified antigen each derived from donors cell lines selected for their expressed DQ types.

Bead Blend– Control Beads3 CON Beads conjugated with: No Antigen (Mock Beads) Human Serum Albumin or Human Platelet Glycoprotein IV3 CON Beads conjugated with: No Antigen (Mock Beads) Human Serum Albumin or Human Platelet Glycoprotein IV
Conjugate ConcentrateSolution containing: Phycoerythrin conjugated goat affinity purified antibody to human IgG in a stabilizer. Contains 0.1% sodium azide.Solution containing: Phycoerythrin conjugated goat affinity purified antibody to human IgG in a stabilizer. Contains 0.1% sodium azide.
Wash BufferPhosphate Buffered Saline containing Bovine Serum Albumin, Detergent, and 0.1% sodium azide, pH 7.1 – 7.3.Phosphate Buffered Saline containing Bovine Serum Albumin, Detergent, and 0.1% sodium azide, pH 7.1 – 7.3.
Positive Control SerumHuman serum containing antibodies to HLA Class II with addition of 0.1% NaN3Human serum containing antibodies to HLA Class II with addition of 0.1% NaN3
Negative Control SerumHuman serum which does not contain any HLA-specific antibodies with addition of 0.1% NaN3Human serum which does not contain any HLA-specific antibodies with addition of 0.1% NaN3

Lifecodes Class II IDv2, Enhanced, is similar to Lifecodes Class II ID in the following:

  1. Lifecodes Class II IDv2 has the same intended use and indications for use compared to the predicate device.
  2. Lifecodes Class II IDv2 utilizes the same technology (a bead based Luminex immunoassay) and assay steps as the predicate device.
  3. Lifecodes Class II IDv2 utilizes the same assay components as the predicate devices.
  4. Lifecodes Class II IDv2 uses the same sample type (serum) as the predicate device.

Lifecodes Class II IDv2 differs from the predicate device in the following:

  1. Lifecodes Class II IDv2 has additional HLA-conjugated beads designed to increase capability of detecting HLA class II antibodies.

VIII. Support of Substantial Equivalence with Performance Data:

The details of each of the following studies are covered in the Performance Studies included in this 510(k). Only a brief summary of these studies is provided in this section.

Assay Reproducibility Studies

Description of Study:
The specific objective of the reproducibility study was to assess between-user and betweensite variability in the performance characteristics of a single lot of LM2Q. The reproducibility study was conducted at three different locations, two external and at Gen-Probe; Waukesha, WI. The sera were selected for their range of antibody reactivity to representative DR antigens and all known DQ antigens.

Results and Analysis
Lifecodes Class II IDv2 (LM2Q) is a qualitative assay and results are reported as positive or negative for HLA class II antibodies. The qualitative result is determined as follows. The LM2Q assay generates a median fluorescence intensity (MFI) value for each bead within the bead blend using the Luminex instrument. The MFI of each bead is then analyzed against the background of the assay based on the control beads included in the bead blend to generate a ratio. From this ratio is subtracted a pre-determined lot-specific background ratio for that bead. Resulting ratios that are above zero are interpreted as positive for that bead. A sample is considered positive if any of the HLA antigen beads have a positive ratio and a pattern of antibody reactivity present.

The qualitative reportable result (Pos/Neg) for each of the seven samples was analyzed for % agreement. The results showed that there was 100% agreement within run (between duplicates) and between run (each day) for each sample. The data also show that there was 100% agreement between each operator and site.

Conclusions:
The Lifecodes Class II IDv2 Assay showed acceptable assay reproducibility of the qualitative reportable results (Pos/Neg).

Lot-to-Lot Repeatability Studies

Description of Study:
The specific objective of the repeatability study was to assess of the reproducibility of assay results between lots. Three lots were included in the study; all testing occurred at one location and performed by one operator. The sera were selected for their range of antibody reactivity to representative DR antigens and all known DQ antigens. Each sample was tested in 20 replicates in 1 testing event with three different lots of LM2Q.

Results and Analysis
The qualitative result for each sample was obtained as described above for the reproducibility study. The qualitative reportable result (Pos/Neg) for each of the seven samples was analyzed for % agreement. The results showed that there was 100% agreement within run and between lots for each sample.

Conclusions:
The Lifecodes Class II IDv2 Assay showed acceptable between lot repeatability of the qualitative reportable results (Pos/Neg).

Comparison of Methods Studies

Description of Studies
Two separate external studies were conducted in which the Lifecodes Class II IDv2 (LM2Q) assay was compared to the Lifecodes Class II ID (LM2) and Lifecreen (LMX) assays. LMX is a qualitative Luminex bead-based immunoassay used to detect IgG to HLA Class I and Class II. In addition to other HLA class I and class II antigens, LMX includes a separate bead to which DQ antigen is conjugated allowing for the detection of DQ-reactive antibodies. The detection of DQ-reactive antibodies with LM2Q was compared to the qualitative results obtained with the DQ-conjugated beads in LMX. Each study site tested 300 previously collected serum samples in the LM2Q, LM2, and LMX assays.

Results and Analysis:
The comparison of LM2Q to LM2 was performed in order to assess total antibody detection as well as DR-reactive antibody detection. The comparison of LM2Q to LMX was performed in order to assess the detection of DQ-reactive antibodies. The data from both sites were combined and analyzed with 2x2 table analysis to calculate copositivity, co-negativity, and % agreement (95% confidence interval).

In the comparison of overall class II-specific antibody detection (LM2Q vs. LM2), the Lifecodes Class II IDv2 kits showed 99.7% co-positivity (98.3-99.9%), 72.2% conegativity (66.6-77.1%), and 87.2% (84.3-89.6%) agreement for 600 sera samples evaluated when compared to results obtained with Lifecodes Class II ID method (95% confidence limits). The values for co-negativity and agreement reflect the enhanced detection of DQ-reactive antibodies with Class II IDv2.

In the comparison of DR-reactive antibody detection (LM2Q vs. LM2 DR beads), the Lifecodes Class II IDv2 kits showed 97.5% co-positivity (95.2-98.7%), 98.2% conegativity (95.9-99.2%), and 97.8% (96.6-98.7%) agreement for 600 sera samples evaluated when compared to results obtained with Lifecodes Class II ID method (95% confidence limits). The values reflect the similar design of LM2Q and LM2: both products incorporate a panel of bead populations conjugated with DR.

In the comparison of DQ-reactive antibody detection (LM2Q vs. LMX DQ beads), the Lifecodes Class II IDv2 kits showed 99.6% co-positivity (98.0-99.9%), 81.9% conegativity (77.3-85.8%), and 90.3% (87.7-92.4%) agreement for 600 sera samples evaluated when compared to results obtained with the Lifescreen Deluxe method (95% confidence limits). Again, the values for co-negativity and agreement reflect the enhanced detection of DQ-reactive antibodies. While some of these DQ-reactive antibodies would be expected to be detected with LMX, because the DQ-conjugated bead population in LMX is conjugated with a pool of DQ antigens and the DQ beads in LM2Q are conjugated with the DQ antigen purified from a single donor, there is greater representation of a given DQ antigen which would increase the likelihood of detection of DQ-reactive antibodies.

Conclusions:
The Lifecodes Class II IDv2 assay showed acceptable co-positivity (sensitivity), conegativity (specificity), and overall agreement with the comparative devices, Lifecodes Class II ID and Lifecodes LifeScreen.

Interfering Substances Testing
Description of Study:
The drugs commonly used in a clinical transplant situation were tested in the original LifeScreen Deluxe assay (BK090002) to determine if their presence was associated with incorrect qualitative reportable results (Pos/Neg). The results of that previous study are applicable to the LIFECODES Class II IDv2 since the technology has not changed.

Results and Analysis:
Thymoglobulin was previously shown to interfere with the assay at a concentration of 10ug/mL. However, none of the drugs listed in the table below showed interference in Lifecodes Lifescreen Deluxe at the concentrations listed.

IDHighest concentration tested
Remicade (infliximab)10μg/mL
Rituxan (rituximab)10μg/mL
Zenapax (daclizumab)10μg/mL
Campath (alemtuzumab)10μg/mL
Gammagard (IVIgG)200μg/mL

Conclusions:
Based on the results obtained with LifeScreen Deluxe and that LIFECODES Class II IDv2 uses Class II HLA-conjugated beads, it is inferred that the Lifecodes Class II IDv2 assay might also yield false positive results in the presence of Thymoglobulin at a concentration of 10μg/mL.

Stability Studies
The stability of the Lifecodes ID Class II IDv2 was determined as an internal study. The studies were carried out for a period of at least 12 months on multiple lots.

Description of Study:
The evaluation included kits taken from 3 different lots: 021809-LM2Q, 072809- LM2Q, 090909-LM2Q. The scope of the testing includes two monoclonals and a panel of allosera selected to have a range of reactivity and strengths to DQ. Data were collected on approximately monthly intervals. The focus of this study was on the performance of the DQ-conjugated beads alone. The DR-conjugated beads are identical with those found in LM2 which has a demonstrated shelf-life of 12 months.

Results and Analysis:
The MFI values were plotted as function of the elapsed time. The data showed a slight decrease in signal over time. A significant decrease in signal (>25%) is not projected to occur until well after 12 months.

The MFI values collected with each serum sample were analyzed to determine the qualitative result (Pos/Neg) for each bead. The data showed that the reportable results determined for each bead were consistent over the course of the study. Beads that were initially positive remained positive; those that were negative remained negative.

Beads which displayed borderline results showed both positive and negative results. These beads continued to display borderline results over the course of the study, neither becoming permanently positive or negative.

Conclusion:
The data support a 12 month shelf life for LIFECODES Class II IDv2.

IX. Conclusion
Based on the evaluation against the predicate device (Lifecodes Class II ID), these data demonstrate that Lifecodes Class II IDv2 performs comparable to the predicate device and the Lifecodes Class II IDv2 kit does not present new issues of safety and effectiveness.