(329 days)
EasySep Human Bone Marrow CD138 Positive Selection Kit is an in vitro diagnostic device intended to enrich CD138+ cells from bone marrow collected from patients diagnosed with multiple myeloma. The CD138+ cells are enriched by immunomagnetic positive selection for use in validated downstream assays. The end-user is responsible for validation of this kit for use with the assay. For in vitro diagnostic use by laboratory professionals.
The EasySep CD138 Human Bone Marrow CD138 Positive Selection Kit contains reagents to enrich CD138 positive (CD138+) human bone marrow cells. The kit contains reagents for up to 60 mL of bone marrow purifications. The kit contents are listed in Table 1. Materials required but not provided are shown in Table 2.
Acceptance Criteria and Device Performance for EasySep Human Bone Marrow CD138 Positive Selection Kit
This document summarizes the acceptance criteria and a detailed account of the studies conducted to demonstrate the EasySep Human Bone Marrow CD138 Positive Selection Kit meets these criteria.
1. Table of Acceptance Criteria and Reported Device Performance
| Parameter | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| Reproducibility with Contrived Samples | Within Lot (across site) CV for Medium/High CD138+ panel members: < 15% Within Lot (across site) CV for Low CD138+ panel members: < 35% Within Site (across lot) CV for Medium/High CD138+ panel members: < 15% Within Site (across lot) CV for Low CD138+ panel members: < 35% | Within Lot (across site) CV: - High (>15%): 4.0% (2.8 - 6.0%) - Med (3-15%): 4.5% (2.2 - 7.0%) - Low (<3%): 11.9% (8.2 - 18.3%) Within Site (across lot) CV: - All medium and high CD138+ panel members: < 15% - All low CD138+ panel members: < 35% Fold Enrichment: - Low CD138+: 85.7-fold (20.2-206.0) - Med CD138+: 9.3-fold (5.0-15.4) - High CD138+: 3.8-fold (2.6-5.5) Purity (Enriched Mean): - Low CD138+: 56.2% - 79.6% - Med CD138+: 85.5% - 92.6% - High CD138+: 88.5% - 94.0% |
| Reproducibility with Clinical Specimens | Normal/Abnormal disposition: Concordant between kit lots for a given FISH probe. | For all 9 BMA tested, the percentage of abnormal cells for each probe were very similar between kit lots and the same normal/abnormal disposition for a given probe was concordant between the pairs of EasySep Human Bone Marrow CD138 Positive Selection Kit lots. |
| Repeatability | Flow Cytometry Analysis (enriched CD138+ purity CV): - Low initial CD138+ frequency: -- Repeatability: N/A (Implicitly met by overall within-laboratory) -- Within-laboratory: < 35% - Medium initial CD138+ frequency: -- Repeatability: N/A (Implicitly met by overall within-laboratory) -- Within-laboratory: < 15% FISH Analysis: All enriched samples for low and medium initial CD138+ frequency meet abnormal FISH patterns. | Flow Cytometry Analysis (enriched CD138+ purity CV): - Low initial CD138+ frequency: -- Repeatability: 0.81% to 1.56% -- Within-laboratory: 0.84% to 1.77% - Medium initial CD138+ frequency: -- Repeatability: 0.17% to 1.41% -- Within-laboratory: 0.65% to 0.96% FISH Analysis: All enriched samples sent for FISH analysis (n=3 per panel member, one replicate per run) gave abnormal FISH patterns. |
| Analytical Specificity (Interference - Excess Heparin) | Enriched CD138+ purity from test samples (with excess heparin) should be > 90% of enriched CD138+ purity from control samples (without excess heparin). Statistical difference: Not statistically different from control specimens (for low frequency specimens). | Three clinical specimens (initial 0.4%, 5.0%, 31.3%): - Specimen #1: 91.0% of control - Specimen #2: 103% of control - Specimen #3: 98.0% of control Three low frequency clinical specimens (initial <0.2%): - Specimen #1: 99% of control - Specimen #2: 100% of control - Specimen #3: 100% of control - P-value for one-tailed t-test: 0.2740 (not statistically different). |
| Specimen Stability (FISH) | All enriched samples should have conclusive FISH results. Each of the five FISH probes should result in the same FISH disposition (normal or abnormal) across time points (baseline, 44-54h, ~73h) when initial CD138 frequencies were above the putative LLoD for the flow assay (0.14%). | Specimen #1 (initial 10.6%): FISH results for three probes analyzed did not change across time points (baseline -> 73h). Specimen #2 (initial 0.5%): FISH results consistent with baseline and 44-54h. More variable (~73h) due to insufficient cells. Specimen #3 (initial 0.4%): FISH disposition for three probes analyzed consistent across time points (baseline -> ~73h). Overall, FISH disposition was consistent between baseline and ~73h for all three specimens when initial CD138 frequencies were above the putative LLoD for the flow assay. |
| Kit Stability (Shelf-Life and In-Use) | Ability to enrich CD138+ cells to a purity of >95% and >70% recovery. | Met all acceptance criteria at long-term and in-use storage (5°C ± 3°C) for up to 7 months, and accelerated storage (25°C ± 2°C/60% ± 5% RH) for up to 7 days. Supports a 6-month shelf life at 5°C ± 3°C. |
| Kit Stability (Shipping) | CD138+ cell purity had to be >95% with at least 70% of cells recovered after exposure to summer and winter temperature profiles for 48 hours. | All acceptance criteria were met at the long-term storage condition of 5°C ± 3°C for up to 7 months, demonstrating product performance unaffected by transport stress. Supports a 6-month shelf-life following transport stress. |
| Limit of Detection (Contrived Samples) | Lowest initial CD138+ frequency that gives consistent abnormal FISH results in a contrived specimen after CD138+ cell enrichment. | Consistently achieved abnormal t(11;14) disposition in FISH analysis for contrived specimens targeting as low as 0.08% initial CD138+ frequency. Final LoD set at 0.05% CD138+ (limit of detection of the flow cytometer). |
| Limit of Detection (Clinical Samples) | Overall enriched purity CV ≤ 35%. Enriched samples' FISH disposition same as undiluted clinical specimen's FISH disposition for all probes. | Specimen enriched from 0.05% to 4.1% - 7.4%. Overall enriched purity CV was 19.7% (meeting ≤35%). All enriched samples had FISH disposition identical to the undiluted clinical specimen (100% abnormal for Chromosome 13q deletion and TP53). |
| Clinical Study (FISH) | Increased percentage of abnormal nuclei in enriched samples compared to unenriched samples. Successful enrichment of a range of bone marrow CD138+ cells (low, intermediate, and high) with viable cells for FISH. | CD138 purity increased from 12.9% ± 15.9% (pre-enrichment) to 79.6% ± 19.9% (post-enrichment). Fold enrichment > 1 for all specimens with CD138 frequencies <40% prior to enrichment. In 14 patients, abnormal nuclei were only detected after enrichment. For all patients, the percentage of abnormal nuclei detected was higher in the enriched sample compared to the unenriched sample. Demonstrates utility for detection of multiple FISH probes. |
2. Sample Size Used for the Test Set and Data Provenance
- Reproducibility with Contrived Samples:
- Test Set: 16 panel members (contrived samples at low, medium, and high CD138+ levels). More panel members (6 each) at low and medium levels, and 4 at the high level.
- Data Provenance: Not explicitly stated, but "healthy donor whole blood" was used as a matrix for spiking, implying healthy human blood products. The study was performed at "three independent sites," suggesting multi-site internal validation rather than external country-specific data. Retrospective/prospective not specified for sample collection, but the study design is prospective.
- Reproducibility with Clinical Specimens:
- Test Set: 9 clinical Multiple Myeloma (MM) bone marrow aspirates (BMA). Each split to test two different kit lots.
- Data Provenance: Multiple Myeloma patient bone marrow aspirates. The document does not specify countries of origin, but it is implied to be clinical samples from patients. A prospective study design for testing these samples is implied.
- Repeatability:
- Test Set: 6 panel members (contrived samples) generated by spiking MM cell line SK-MM-2 into healthy donor bone marrow. Two panel members (one low, one medium frequency) were generated each day over 3 non-consecutive days, resulting in 6 unique panel members for the study. Each operator performed 3 runs of enrichments in quadruplicate (n=12 total replicates per panel member for flow cytometry, n=3 selected replicates for FISH).
- Data Provenance: "Healthy donor bone marrow" and "CD138+ cell line SK-MM-2." Similar to reproducibility, likely internal multi-site data. Prospective study design.
- Analytical Specificity (Interference - Excess Heparin):
- Test Set (Study 1): 3 fresh clinical multiple myeloma bone marrow specimens.
- Test Set (Study 2, for low frequency specimens): 3 clinical specimens prepared by adding MM bone marrow mononuclear cells into healthy donor bone marrow at <0.2% initial CD138 frequency.
- Data Provenance: Clinical Multiple Myeloma bone marrow specimens and spiked healthy donor bone marrow. Country of origin not specified. Prospective study design.
- Specimen Stability:
- Test Set: ~six MM patient BMAs. Data is shown for 3 specimens specifically.
- Data Provenance: Multiple Myeloma patient bone marrow aspirates. Country of origin not specified. Prospective study design for testing.
- Kit Stability (Shelf-Life and In-Use, Shipping):
- Test Set: Three kit lots for shelf-life, one lot for in-use stability, one batch for shipping stability.
- Data Provenance: Internal testing of kit components. Prospective study design.
- Limit of Detection (Contrived Samples):
- Test Set: Whole Blood (WB) or Bone Marrow (BM) samples from healthy donors spiked with SK-MM-2 cell line at 9 different serially diluted target percentages (from ~20% down to 0%). Each sample enriched in duplicate.
- Data Provenance: Healthy donor WB or BM and CD138+ cell line SK-MM-2. Likely internal multi-site data. Prospective study design.
- Limit of Detection (Clinical Multiple Myeloma Bone Marrow Specimens):
- Test Set (Study 1): 5 low CD138 frequency clinical specimens (pre-enrichment frequencies 0.2%, 0.5%, 0.7%, 1.5%, 1.7%).
- Test Set (Study 2, Verification): 1 clinical specimen produced by spiking MM patient BMA into healthy donor BMA at 0.05% initial CD138 frequency. Split into 6 fractions (2 kit lots, 3 replicates per lot).
- Data Provenance: Clinical Multiple Myeloma bone marrow specimens and spiked healthy donor BMA. Country of origin not specified. Prospective study design.
- Clinical Study:
- Test Set: 33 Multiple Myeloma patient bone marrow aspirates.
- Data Provenance: Multiple Myeloma patient bone marrow aspirates. The document refers to "two studies" where these samples were collected but doesn't specify if they were from multiple countries or regions. Prospective analysis of these collected samples.
3. Number of Experts Used to Establish Ground Truth and Qualifications
- For all studies involving FISH analysis: "Specimen slides were enumerated by a minimum of two readers."
- Qualifications of Experts: The document does not explicitly state the qualifications of the "readers" who enumerated the FISH slides (e.g., "radiologist with 10 years of experience"). Based on the context of FISH analysis, these would typically be trained cytogenetic technologists or pathologists with expertise in FISH interpretation.
4. Adjudication Method for the Test Set
- For FISH analysis in the Clinical Study and LoD studies: "Specimen slides were enumerated by a minimum of two readers; 50 nuclei per reader were analyzed, for a total of 100 nuclei. If counts for a reader were below or equal to the cutoff corresponding to each probe, then the result from the reader was determined to be normal and the percentage of abnormal nuclei would be indicated as '0'."
- This suggests a form of consensus or independent reading with a pre-defined rule for determining "normal" based on individual reader counts. It is not explicitly stated if there was an adjudication procedure for discordant readings between the two readers, or if a third reader was involved (e.g., 2+1 method).
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
- No, an MRMC comparative effectiveness study was not performed as described. The studies focus on the analytical and clinical performance of the device itself (enrichment efficacy, reproducibility, LoD) rather than comparing human reader performance with and without AI assistance. The device is a "Hematopoietic cell enrichment kit," meaning it's a tool for sample preparation, not an interpretive AI algorithm intended to assist human readers directly.
6. Standalone Performance
- Yes, a standalone performance was done for the device's function. The entire document details the performance of the "EasySep Human Bone Marrow CD138 Positive Selection Kit" in enriching CD138+ cells. This is the standalone performance of the device (a physical kit for cell separation), not an algorithm. The device's output (enriched cells) is then used in downstream assays (flow cytometry, FISH), which are performed by humans and/or other laboratory instruments. The performance metrics reported (purity, fold enrichment, CV, FISH disposition) are directly attributable to the standalone function of the kit.
7. Type of Ground Truth Used
- Flow Cytometry: For quantification of CD138+ cell purity and initial frequencies. This is a widely accepted and quantitative method in immunology.
- FISH Analysis: For detecting chromosomal abnormalities. This is a standard cytogenetic method, with "normal/abnormal disposition" and "percentage of abnormal nuclei" serving as the ground truth for genetic status, based on enumeration by expert readers (as described in section 3).
- Contrived Samples: For reproducibility and LoD studies, the "ground truth" for the initial CD138+ frequency was established by spiking a known cell line (SK-MM-2) into healthy donor blood/bone marrow. The t(11;14) translocation characteristic of SK-MM-2 cells then serves as the ground truth for "abnormal" FISH results in these contrived samples.
- Clinical Specimens: For analytical specificity and clinical studies, the "ground truth" for abnormal FISH patterns (e.g., 13q deletion, t(11;14)) comes from the FISH analysis itself, which is a gold standard for detecting these abnormalities in patient samples, as enumerated by expert readers.
8. Sample Size for the Training Set
- Not applicable. This device is a physical kit for cell enrichment, not a machine learning model that requires a "training set." The studies performed are verification and validation studies to demonstrate the kit's performance characteristics.
9. How the Ground Truth for the Training Set Was Established
- Not applicable. As the device is not an AI/ML algorithm, there is no "training set" or corresponding ground truth to establish for it in the context of machine learning. The studies described are for analytical and clinical validation of a physical diagnostic device.
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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR EasySep Human Bone Marrow CD138 Positive Selection Kit DECISION SUMMARY
I Background Information:
A De Novo Number
B Applicant
STEMCELL Technologies Canada Inc.
C Proprietary and Established Names
EasySep™ Human Bone Marrow CD138 Positive Selection Kit
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
|---|---|---|---|
| QYO | Hematopoietic cellenrichment kit | 21 CFR 866.6120 | Immunology |
II Submission/Device Overview:
A Purpose for Submission:
De Novo request for evaluation of automatic class III designation for EasySep Human Bone Marrow CD138 Positive Selection
B Measurand:
CD138 (Syndecan-1)
C Type of Test:
Tool for selection of human bone marrow CD138+ plasma cells
III Indications for Use:
A Intended Use(s):
See Indications for Use below.
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B Indication(s) for Use:
EasySep Human Bone Marrow CD138 Positive Selection Kit is an in vitro diagnostic device intended to enrich CD138+ cells from bone marrow collected from patients diagnosed with multiple myeloma. The CD138+ cells are enriched by immunomagnetic positive selection for use in validated downstream assays. The end-user is responsible for validation of this kit for use with the assay. For in vitro diagnostic use by laboratory professionals.
C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only For in vitro diagnostic use
D Special Instrument Requirements:
Not applicable: The EasySep Human Bone Marrow CD138 Positive Selection Kit was validated for manual use.
IV Device/System Characteristics:
A Device Description:
The EasySep CD138 Human Bone Marrow CD138 Positive Selection Kit contains reagents to enrich CD138 positive (CD138+) human bone marrow cells. The kit contains reagents for up to 60 mL of bone marrow purifications. The kit contents are listed in Table 1. Materials required but not provided are shown in Table 2.
| Component | PackagingConfiguration | Storage | Content |
|---|---|---|---|
| EasySep Human BoneMarrow CD138 PositiveSelection Cocktail(300-0394) | 3 x 1 mL vials | Store at 2 -8°C. Donot freeze. | A combination of monoclonal antibodies inDulbecco's Phosphate Buffered Saline. Includes an Fcreceptor blocking antibody and Recombinant HumanAlbumin stabilizer. |
| EasySep DextranRapidSpheres 50105(300-0400) | 3 x 1 mL vials | Store at 2 -8°C. Donot freeze. | A suspension of magnetic particles in water. |
Table 1. EasySep Human Bone Marrow CD138 Positive Selection Kit Components
Table 2. Materials required but not provided (Sold separately)
| Component | Configuration | Storage | Content |
|---|---|---|---|
| EasySep Red Blood CellLysis Buffer, 10xConcentrate (100-0749) | 1 x 20 mLBottle | Store at15 - 25°C. | A 10x concentrated red blood cell lysisreagent. |
| EasySep Buffer(100-0780) | 1 x 1 L | Store at2 - 8°C. Do notfreeze. | A Dulbecco's Phosphate Buffered Salinebased cell separation buffer. |
| "The Big Easy" EasySepMagnet (18001) | 1 | Ambient temperature | Immunomagnetic column-free magnet. |
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B Principle of Operation
The EasySep Human Bone Marrow CD138 Positive Selection Kit is designed to enrich plasma cells expressing the CD138 surface antigen from fresh human whole bone marrow aspirates. Plasma cells are targeted with antibody complexes that crosslink CD138+ cells to dextran-coated magnetic particles. When the bone marrow specimen is placed into an EasySep magnet. CD138+ cells linked to magnetic particles are retained by the magnetic field while CD138- cells remain in suspension. This allows the CD138 negative (CD138-) cell fraction to be removed by pouring off the suspension containing unlabeled, unbound cells. Enriched CD138+ cells are collected by the removal of the sample from the magnet and can then be used in downstream applications.
V Standards/Guidance Documents Referenced:
CLSI Guideline EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures
CLSI Guideline EP07-A2: Interference Testing in Clinical Chemistry
CLSI Guideline EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents
VI Performance Characteristics:
A Analytical Performance:
-
- Reproducibility:
a) Reproducibility with Contrived Samples
A study was performed to demonstrate the reproducibility of the enrichment process. Due to challenges with having adequate sample for three sites, a study was performed using contrived samples generated by spiking a multiple myeloma cell line into healthy donor whole blood each day at 3 CD138+ levels based on flow cytometry events (low, medium, and high representing <3%. 3-15%. and >15%. respectively) to create 16 panel members. More panel members were studied at the low and medium levels (6 versus 4 for the high level) to demonstrate enrichment at the more challenging levels. Reproducibility was evaluated at three sites by two operators at each site for 8 non-consecutive days. Each of the three independent sites received six aliguots (from 16 panel members) from the same panel members. Each operator performed enrichments in duplicate using three different reagent lots on each panel member. After the enrichments were completed. all samples were given to a single operator to perform flow cytometry of the enriched and unenriched samples to measure CD138+ cell purity. Two different CD138 frequency contrived specimens were prepared for enrichments on each day.
Table 3 shows a statistical summary of the overall, site-to-site, lot-to-lot variability, and fold-enrichment from the high, medium, and low CD138+ panel members. Enrichment increased a mean of 85.7-fold in the low CD138+ initial frequency samples, 9.3-fold in the CD138+ medium frequency samples, and 3.8-fold in the high starting samples.
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| CD138+ InitialFrequency | N | RepeatabilityCV (%) | Between SiteCV (%) | Between Lot CV(%) | ReproducibilityCV (%) | Fold Enrichment |
|---|---|---|---|---|---|---|
| Mean (Min. - Max) | ||||||
| High (> 15%) | 4 | 0.7 (0.5 - 0.9) | 4.0 (2.8 - 5.9) | 0.9 (0.5 - 1.6) | 4.0 (2.8 - 6.0) | 3.8 (2.6 - 5.5) |
| Med (3 - 15%) | 6 | 1.6 (0.8 - 3.0) | 4.2 (2.1 - 6.8) | 2.3 (1.0 - 3.9) | 4.5 (2.2 - 7.0) | 9.3 (5.0 - 15.4) |
| Low (< 3%) | 6 | 5.4 (3.5 - 8.9) | 9.7 (5.5 - 13.8) | 8.6 (4.5 - 14.2) | 11.9 (8.2 - 18.3) | 85.7 (20.2 - 206.0) |
Table 3. EasySep CD138 Reproducibility Study Variability and Fold Enrichment Summary
Table 4 shows the mean plus minimum and maximum CD138+ purity for unenriched and enriched samples from all panel members across the three study sites. Low CD138+ panel members had mean CD138+ cell purities ranging from 0.7% - 1.5% before enrichment and increased to mean CD138+ cell purities of 56.2% - 79.6% after EasySep enrichment. Medium CD138+ panel members had mean CD138+ cell purities ranging from 7.0% - 12.3% before enrichment and increased to mean CD138+ cell purities of 85.5% - 92.6% after EasySep enrichment. High CD138+ panel members had mean CD138+ cell purities ranging from 17.9% - 22.8% before enrichment and increased to mean CD138+ cell purities of 88.5% - 94.0% after EasySep enrichment.
| Target Freq | Panel Member | Unenriched Purity | Enriched Purity | ||||
|---|---|---|---|---|---|---|---|
| Mean | Min | Max | Mean | Min | Max | ||
| Low | 1 | 1.5 | 0.8 | 2 | 56.6 | 43.1 | 74.9 |
| 5 | 1.3 | 0.7 | 2 | 73 | 62.3 | 86.7 | |
| 8 | 0.7 | 0.6 | 0.8 | 56.2 | 43.8 | 66.5 | |
| 12 | 0.7 | 0.4 | 0.9 | 76.9 | 66.4 | 89.3 | |
| 14 | 1.2 | 0.6 | 1.8 | 79.6 | 71.4 | 91.2 | |
| 15 | 0.8 | 0.4 | 1.2 | 74.9 | 57.9 | 82.8 | |
| Med | 2 | 9.4 | 7.2 | 11.3 | 89.8 | 79.6 | 95.3 |
| 3 | 9.2 | 7 | 12.5 | 88.9 | 84.3 | 93 | |
| 7 | 7.3 | 5.3 | 9.9 | 86.3 | 81.5 | 90.4 | |
| 10 | 7 | 5.5 | 8.3 | 86.4 | 75.3 | 93.2 | |
| 13 | 12.3 | 11.1 | 14.3 | 92.6 | 89.7 | 95.4 | |
| 16 | 10.4 | 6.2 | 14.6 | 85.5 | 78.7 | 90.4 | |
| High | 4 | 19 | 16.4 | 21.3 | 94 | 89.9 | 96.8 |
| 6 | 22.8 | 19.3 | 26.6 | 92.3 | 87.8 | 96.4 | |
| 9 | 18 | 17 | 18.6 | 88.5 | 82.9 | 95.2 | |
| 11 | 17.9 | 14.3 | 20 | 93.2 | 90.3 | 96.3 |
Table 4. Unenriched and Enriched CD138+ Cell Purity for Low, Medium, and High CD138+ Panel Members as Determined by FACS Analysis by All Three Study Sites
Separate analyses of variance were performed within each of the three sites and the three kit lots. Table 5 shows analysis results by kit lot and panel member, with precision estimates for repeatability and within lot/between sites. Similarly, Table 6 shows analysis results by site and panel member, with precision estimates for repeatability and within site/between lots. In both sets of analyses, all medium and high CD138+ panel members within site and lot had CV < 15%. Similarly, all low CD138+ panel members within site and lot had CV < 35%, which met the acceptance criteria due to the expected higher variability for low CD138+ starting samples. Thus, the variability is well-controlled within lot (across site) and within site (across lot).
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| Table 5. Precision Estimates by Kit Lot | |||||||
|---|---|---|---|---|---|---|---|
| Kit Lot | PanelMember | Mean StartPurity* | Mean EnrichedPurity** | Repeatability | Within Lot | ||
| 1 | 8 | 0.66 | 60.17 | 2.13 | 3.5 | 5.01 | 8.3 |
| 12 | 0.71 | 72.48 | 1.21 | 1.7 | 8.39 | 11.6 | |
| 15 | 0.82 | 68.15 | 4.93 | 7.2 | 6.23 | 9.1 | |
| 14 | 1.19 | 77.56 | 1.71 | 2.2 | 4.56 | 5.9 | |
| 5 | 1.26 | 75.47 | 0.38 | 0.5 | 9.86 | 13.1 | |
| 1 | 1.47 | 55.66 | 7.13 | 12.8 | 7.18 | 12.9 | |
| 10 | 6.96 | 87.05 | 3.4 | 3.9 | 5.22 | 6 | |
| 7 | 7.27 | 87.26 | 0.33 | 0.4 | 1.17 | 1.3 | |
| 3 | 9.19 | 88.02 | 1.86 | 2.1 | 2.42 | 2.8 | |
| 2 | 9.44 | 88.91 | 0.65 | 0.7 | 7.47 | 8.4 | |
| 16 | 10.44 | 85.49 | 1.38 | 1.6 | 4.93 | 5.8 | |
| 13 | 12.34 | 92.23 | 0.71 | 0.8 | 2.1 | 2.3 | |
| 11 | 17.9 | 93.32 | 0.14 | 0.2 | 2.49 | 2.7 | |
| 9 | 18.01 | 87.11 | 1.13 | 1.3 | 5.02 | 5.8 | |
| 4 | 18.99 | 93.66 | 0.35 | 0.4 | 2.94 | 3.1 | |
| 6 | 22.84 | 92 | 0.45 | 0.5 | 3.99 | 4.3 | |
| 2 | 8 | 0.66 | 55.67 | 1.68 | 3 | 9.79 | 17.6 |
| 12 | 0.71 | 80.84 | 1.96 | 2.4 | 5.2 | 6.4 | |
| 15 | 0.82 | 79.59 | 1.14 | 1.4 | 1.96 | 2.5 | |
| 14 | 1.19 | 84.84 | 2.08 | 2.4 | 6.17 | 7.3 | |
| 5 | 1.26 | 72.02 | 2.72 | 3.8 | 6.6 | 9.2 | |
| 1 | 1.47 | 64.12 | 2.93 | 4.6 | 9.24 | 14.4 | |
| 10 | 6.96 | 88.4 | 2.54 | 2.9 | 4.74 | 5.4 | |
| 7 | 7.27 | 87.78 | 0.56 | 0.6 | 1.93 | 2.2 | |
| 3 | 9.19 | 90.19 | 0.85 | 0.9 | 2.19 | 2.4 | |
| 2 | 9.44 | 91.62 | 0.84 | 0.9 | 4.65 | 5.1 | |
| 16 | 10.44 | 84.9 | 0.96 | 1.1 | 3.91 | 4.6 | |
| 13 | 12.34 | 93.26 | 1.01 | 1.1 | 2.1 | 2.3 | |
| 11 | 17.9 | 93.36 | 0.78 | 0.8 | 2.54 | 2.7 | |
| 9 | 18.01 | 88.89 | 0.32 | 0.4 | 5.65 | 6.4 | |
| 4 | 18.99 | 94.27 | 0.72 | 0.8 | 3.12 | 3.3 | |
| 6 | 22.84 | 92.08 | 0.38 | 0.4 | 3.96 | 4.3 | |
| 3 | 8 | 0.66 | 52.7 | 3.68 | 7 | 6.54 | 12.4 |
| 12 | 0.71 | 77.45 | 3.58 | 4.6 | 7.67 | 9.9 | |
| 15 | 0.82 | 77.05 | 1.55 | 2 | 3.22 | 4.2 | |
| 14 | 1.19 | 76.55 | 2.44 | 3.2 | 4.08 | 5.3 | |
| 5 | 1.26 | 71.49 | 1.95 | 2.7 | 6.85 | 9.6 | |
| 1 | 1.47 | 50.17 | 1.73 | 3.5 | 6.66 | 13.3 | |
| 10 | 6.96 | 83.71 | 1.16 | 1.4 | 6.84 | 8.2 | |
| 7 | 7.27 | 83.86 | 0.93 | 1.1 | 2.56 | 3.1 | |
| 3 | 9.19 | 88.37 | 0.38 | 0.4 | 3.2 | 3.6 | |
| 2 | 9.44 | 88.72 | 0.69 | 0.8 | 5.9 | 6.7 | |
| 16 | 10.44 | 86.19 | 1.18 | 1.4 | 2.4 | 2.8 | |
| 13 | 12.34 | 92.18 | 0.81 | 0.9 | 1.68 | 1.8 | |
| 11 | 17.9 | 92.97 | 0.26 | 0.3 | 2.7 | 2.9 | |
| Kit Lot | PanelMember | Mean StartPurity* | Mean EnrichedPurity** | Repeatability | Within Lot | ||
| SD | CV (%) | SD | CV (%) | ||||
| 4 | 18.99 | 94.11 | 0.39 | 0.4 | 2.84 | 3 | |
| 6 | 22.84 | 92.88 | 0.86 | 0.9 | 2.81 | 3 |
Table 5. Precision Estimates by Kit Lot
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Start purity = unenriched CD138+ cell purity
*Starting purity for the same sample is averaged across differing measurements from the three sites.
**Mean enriched purity is the average purity of enriched samples across three study sites.
| Panel | Start | Mean Enriched | Repeatability | Within Site | |||
|---|---|---|---|---|---|---|---|
| Site | Member | Purity* | Purity** | SD | CV (%) | SD | CV (%) |
| 1 | 8 | 0.57 | 60.75 | 2.69 | 4.4 | 3.81 | 6.3 |
| 1 | 12 | 0.79 | 84.73 | 2.98 | 3.5 | 3.13 | 3.7 |
| 1 | 15 | 0.92 | 76.93 | 0.72 | 0.9 | 2.73 | 3.5 |
| 1 | 14 | 1.19 | 76.97 | 2.55 | 3.3 | 3.5 | 4.5 |
| 1 | 5 | 0.65 | 81.18 | 1.08 | 1.3 | 4.6 | 5.7 |
| 1 | 1 | 1.58 | 64.33 | 6.66 | 10.4 | 10.24 | 15.9 |
| 1 | 10 | 7.11 | 87.67 | 0.43 | 0.5 | 2.2 | 2.5 |
| 1 | 7 | 6.59 | 85.89 | 0.71 | 0.8 | 2.2 | 2.6 |
| 1 | 3 | 8.06 | 89.44 | 0.94 | 1.1 | 1.6 | 1.8 |
| 1 | 2 | 9.83 | 93.35 | 0.49 | 0.5 | 0.86 | 0.9 |
| 1 | 16 | 10.52 | 85.13 | 0.59 | 0.7 | 1.12 | 1.3 |
| 1 | 13 | 11.13 | 92.7 | 0.47 | 0.5 | 0.62 | 0.7 |
| 1 | 11 | 19.39 | 92.4 | 0.14 | 0.1 | 0.35 | 0.4 |
| 1 | 9 | 17.03 | 86.78 | 0.9 | 1 | 1.22 | 1.4 |
| 1 | 4 | 19.26 | 96.03 | 0.34 | 0.4 | 0.48 | 0.5 |
| 1 | 6 | 22.61 | 92.49 | 0.35 | 0.4 | 0.73 | 0.8 |
| 2 | 8 | 0.6 | 50.2 | 2.97 | 5.9 | 5.51 | 11 |
| 2 | 12 | 0.4 | 73.25 | 2.41 | 3.3 | 6.6 | 9 |
| 2 | 15 | 0.4 | 75.6 | 1.2 | 1.6 | 7.93 | 10.5 |
| 2 | 14 | 0.6 | 77.93 | 0.88 | 1.1 | 6.25 | 8 |
| 2 | 5 | 1.1 | 71.23 | 0.85 | 1.2 | 2.31 | 3.2 |
| 2 | 1 | 0.8 | 54.1 | 2.59 | 4.8 | 5.16 | 9.5 |
| 2 | 10 | 5.5 | 80.72 | 3.9 | 4.8 | 4.91 | 6.1 |
| 2 | 7 | 5.3 | 84.77 | 0.26 | 0.3 | 2.77 | 3.3 |
| 2 | 3 | 7 | 86.23 | 0.52 | 0.6 | 1.65 | 1.9 |
| 2 | 2 | 7.2 | 82.85 | 0.78 | 0.9 | 3.15 | 3.8 |
| 2 | 16 | 6.2 | 82.32 | 1.85 | 2.3 | 3.08 | 3.7 |
| 2 | 13 | 11.6 | 90.63 | 1.23 | 1.4 | 1.23 | 1.4 |
| 2 | 11 | 14.3 | 91.18 | 0.8 | 0.9 | 0.8 | 0.9 |
| 2 | 9 | 18.6 | 84.37 | 0.31 | 0.4 | 1.37 | 1.6 |
| 2 | 4 | 16.4 | 90.63 | 0.82 | 0.9 | 0.82 | 0.9 |
| 2 | 6 | 19.3 | 88.68 | 0.89 | 1 | 1.27 | 1.4 |
| 3 | 8 | 0.82 | 57.58 | 2.19 | 3.8 | 8.56 | 14.9 |
| 3 | 12 | 0.94 | 72.78 | 1.86 | 2.6 | 4.31 | 5.9 |
| 3 | 15 | 1.15 | 72.25 | 5.1 | 7.1 | 8.58 | 11.9 |
| 3 | 14 | 1.79 | 84.05 | 2.43 | 2.9 | 6.43 | 7.7 |
| 3 | 5 | 2.04 | 66.57 | 3.08 | 4.6 | 3.08 | 4.6 |
| 3 | 1 | 2.03 | 51.52 | 3.38 | 6.6 | 7.76 | 15.1 |
| 3 | 10 | 8.28 | 90.77 | 2 | 2.2 | 2.36 | 2.6 |
| 3 | 7 | 9.93 | 88.23 | 0.85 | 1 | 1.83 | 2.1 |
Table 6. Precision Estimates by Site
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| Site | PanelMember | StartPurity* | Mean EnrichedPurity** | Repeatability | Within Site | ||
|---|---|---|---|---|---|---|---|
| 3 | 12.5 | 90.9 | 1.78 | 2 | 1.78 | 2 | |
| 2 | 11.3 | 93.05 | 0.87 | 0.9 | 1.53 | 1.6 | |
| 16 | 14.6 | 89.13 | 0.67 | 0.7 | 0.78 | 0.9 | |
| 13 | 14.3 | 94.33 | 0.67 | 0.7 | 0.93 | 1 | |
| 11 | 20 | 96.08 | 0.2 | 0.2 | 0.2 | 0.2 | |
| 9 | 18.4 | 94.28 | 1.03 | 1.1 | 1.59 | 1.7 | |
| 4 | 21.3 | 95.37 | 0.1 | 0.1 | 0.37 | 0.4 | |
| 6 | 26.6 | 95.78 | 0.41 | 0.4 | 0.44 | 0.5 |
Start purity = unenriched CD138+ cell purity
*Starting purity for the same sample is averaged across all three sites.
** Mean enriched purity is the average purity of enriched samples across three kit lots.
The CD138 EasySep Reproducibility study showed the EasySep Human Bone Marrow CD138 Positive Selection Kit produced consistent enrichment when starting with a specimen of given volume and initial CD138+ cell frequency across three independent laboratories and three separately manufactured kit lots. The precision statistical analysis of all 16 panel members showed that the CD138 cell enrichment performance across three independent laboratories using three independent kit lots gave fold enrichment and overall variability passing the acceptance criteria, demonstrating reproducibility across sites and kit lots.
b) Reproducibility with Clinical Specimens
A study was performed to demonstrate the reproducibility of the enrichment process using a validated FISH assay. Three (3) lots of the EasySep Human Bone Marrow CD138 Positive Selection Kit were tested on multiple myeloma (MM) patient bone marrow aspirates (BMA) in duplicate for performance in downstream FISH assays (Table 7). A total of 9 clinical MM BMA were tested, where each specimen was split in half to test enrichment with two different EasySep Human Bone Marrow CD138 Positive Selection Kit lots. The percentage of abnormal nuclei were analyzed using 5 probes which detect common MM chromosomal abnormalities, including the t(11:14) translocation (CCND1/IGH XT), chromosomes 5, 9, and 15 aneusomies (D5S23, D5S72, CEP 9, CEP 15) and 13g14 deletion.
| Specimen | Lot | Specimen | Lot |
|---|---|---|---|
| MM Specimen 1 | Lot 1 | MM Specimen 6 | Lot 2 |
| Lot 2 | Lot 3 | ||
| MM Specimen 2 | Lot 1 | MM Specimen 7 | Lot 1 |
| Lot 3 | Lot 2 | ||
| MM Specimen 3 | Lot 2 | MM Specimen 8 | Lot 1 |
| Lot 3 | Lot 3 | ||
| MM Specimen 4 | Lot 1 | MM Specimen 9 | Lot 2 |
| Lot 2 | Lot 3 | ||
| MM Specimen 5 | Lot 1 | ||
| Lot 3 |
Table 7. Lot-to-Lot Combinations
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The percentage of cells with an abnormal FISH signal pattern(s) for each probe are listed in Table 8 are shown in bold. Three (3) BMA gave normal FISH signal patterns for all five probes tested. One BMA was abnormal for the t(11:14) translocation and 4 BMA had abnormal signal patterns for the 13q deletion. In all nine BMA tested, the percentage of abnormal cells for each probe were very similar between the plasma cells enriched with the two EasySep Human Bone Marrow CD138 Positive Selection Kit lots. The same normal/abnormal disposition for a given probe was concordant between the pairs of EasySep Human Bone Marrow CD138 Positive Selection Kit lots. Cells which are bold in Table 8 were dispositioned as abnormal with abnormal FISH signal patterns. Cells which are not bold were dispositioned as normal with abnormal FISH signal patterns that did not exceed the cutoffs for percentage of abnormal nuclei. In all cases, normal/abnormal disposition agreed, indicating that the EasySep Human Bone Marrow CD138 Positive Selection Kit is able to produce reproducible FISH results when used on the intended use specimen.
| Table 8. FISH Enumeration Results Using Five Probes on MM BMAs Following Enrichment |
|---|
| Using the EasySep Human Bone Marrow CD138 Positive Selection Kit |
| SampleNo. | Kit Lot | t(11;14)% Abnormal | Trisomy | Tetrasomy | Monosomy | 13qDeletion | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Chr.5 | Chr.9 | Chr.15 | Chr.5 | Chr.9 | Chr.15 | Chr.5 | Chr.9 | Chr.15 | ||||
| 1 | 1 | 0 | 25.5 | 14 | 4.5 | 6.5 | 1 | 1 | 0.5 | 2 | 3.5 | 4 |
| 1 | 2 | 0 | 27 | 18 | 6.5 | 1.5 | 2 | 0.5 | 1.5 | 1 | 1 | 5 |
| 2 | 1 | 0 | 0 | 88 | 0 | 0 | 1.5 | 0 | 0 | 0 | 10 | 96 |
| 2 | 3 | 0 | 1 | 87.5 | 0 | 0 | 0.5 | 0.5 | 1.5 | 0 | 7.5 | 95.5 |
| 3 | 2 | 0 | 74.5 | 55.5 | 49 | 0.5 | 0 | 0 | 0 | 0 | 0 | 84.5 |
| 3 | 3 | 0 | 72 | 59 | 46.5 | 0 | 2.5 | 1 | 0 | 0 | 0 | 80 |
| 4 | 1 | 0 | 2 | 1 | 1.5 | 0 | 0 | 0 | 2.5 | 2.5 | 1 | 2 |
| 4 | 2 | 0 | 3 | 3 | 1 | 0 | 0 | 0 | 1.5 | 4 | 5.5 | 2.5 |
| 5 | 1 | 0 | 9.5 | 5.5 | 6.5 | 0.5 | 1 | 1 | 6 | 2.5 | 5 | 2.5 |
| 5 | 3 | 0 | 3 | 4.5 | 2 | 0.5 | 0.5 | 0.5 | 2 | 2 | 4 | 2.5 |
| 6 | 2 | 0 | 2 | 0 | 53 | 0 | 0 | 0 | 0.5 | 0 | 1 | 96.5 |
| 6 | 3 | 0 | 0.5 | 1.5 | 38.5 | 0 | 1 | 0 | 0.5 | 2 | 3 | 95.5 |
| 7 | 1 | 89 | 0 | 2 | 2 | 0 | 0 | 0 | 0 | 5.5 | 4 | 90.5 |
| 7 | 2 | 90 | 2.5 | 7 | 2 | 0 | 0 | 0.5 | 2 | 1 | 4 | 91 |
| 8 | 1 | 0 | 0.5 | 1.5 | 1 | 0 | 1 | 0 | 2 | 1.5 | 4 | 1 |
| 8 | 3 | 0 | 1.5 | 0.5 | 1 | 2.5 | 0 | 0 | 1.5 | 1 | 3 | 0.5 |
| 9 | 2 | 0 | 63 | 53 | 35 | 5 | 1.5 | 4.5 | 1.5 | 1 | 0 | 2 |
| 9 | 3 | 0 | 60 | 48.5 | 38.5 | 1 | 0 | 0 | 1 | 1.5 | 3.5 | 1 |
2. Repeatability
In this precision study, one operator enriched a low or medium initial CD138+ frequency panel member using one EasySep Human Bone Marrow CD138 Positive Selection Kit lot in quadruplicate over three runs using contrived samples. On three non-consecutive days, a bone marrow specimen from a healthy donor was spiked with the CD138+ cell line SK-MM-2 at a low (< 3%) and a medium (3 - 15%) frequency to generate two separate contrived sample panel members. Two operators performed EasySep enrichments on each of the study days, with each operator enriching from one of the two panel members. Each operator performed three runs of enrichments in quadruplicate on one panel member using one GMP kit lot.
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Table 9 shows the unenriched and enriched CD138+ cell purity from all panel members and enriched CD138+ cell purity from all panel members across all three runs. Low initial CD138+ frequency panel members had mean CD138+ cell purities ranging from 1.32 - 1.51% before enrichment and increased to CD138+ cell purities of 80.99% -87.93% after EasySep enrichment. Medium initial CD138+ frequency panel members had CD138+ cell purities ranging from 9.84% - 10.84% before enrichment and increased to mean CD138+ cell purities of 91.10% - 96.65% after EasySep enrichment.
| Table 9. Unenriched and Enriched CD138+ Cell Purity for Low and Medium Initial CD138+ |
|---|
| Frequency Panel Members as Determined by Flow Cvtometry Analysis |
| Initial CD138+Frequency Category | PanelMember | Unenriched Purity*(% CD138+) | Enriched Purity (%CD138+) | |||||
|---|---|---|---|---|---|---|---|---|
| Low | B1 | 1.51 | 84.91 | 83.05 | 87.48 | 1.51 | 1.77 | 12 |
| Low | B4 | 1.32 | 86.77 | 85.65 | 87.93 | 0.73 | 0.84 | 12 |
| Low | B5 | 1.37 | 82.41 | 80.99 | 83.77 | 0.93 | 1.13 | 12 |
| Medium | B2 | 10.84 | 93.82 | 92.75 | 95.62 | 0.90 | 0.96 | 12 |
| Medium | B3 | 9.84 | 95.70 | 94.81 | 96.65 | 0.62 | 0.65 | 12 |
| Medium | B6 | 10.17 | 93.32 | 91.10 | 94.21 | 0.77 | 0.83 | 12 |
Table 10 shows the CD138+ cell recovery for low and medium initial CD138+ frequency panel members and the corresponding statistical analyses. Low initial CD138+ frequency panel members had CD138+ cell recoveries ranging from 34.98% - 76.69% (Mean = 53.42%). Medium initial CD138+ frequency panel members had CD138+ cell recoveries ranging from 36.89% - 65.16% (Mean = 50.62%). The overall CD138+ cell recovery within-laboratory precision estimates (%CV across three runs with four replicates. n = 12 total) ranged from 5.52% - 27.4% and 10.53% - 18.32% for low and medium initial CD138+ frequency panel members respectively.
Table 10. CD138+ Cell Recovery for Low and Medium Initial CD138 Frequency Panel Members
| Initial CD138Frequency Category | PanelMember | Unenriched Purity*(% CD138+) | CD138+ Cell Recovery (%) | |||||
|---|---|---|---|---|---|---|---|---|
| Low | B1 | 1.51 | 55.30 | 34.98 | 76.69 | 15.15 | 27.4 | 12 |
| Low | B4 | 1.32 | 48.88 | 36.63 | 64.37 | 7.92 | 16.2 | 12 |
| Low | B5 | 1.37 | 56.09 | 50.56 | 63.00 | 3.09 | 5.52 | 12 |
| Medium | B2 | 10.84 | 50.78 | 37.58 | 58.57 | 6.64 | 13.07 | 12 |
| Medium | B3 | 9.84 | 49.04 | 36.89 | 57.99 | 5.16 | 10.53 | 12 |
| Medium | B6 | 10.17 | 52.04 | 37.88 | 65.16 | 9.53 | 18.32 | 12 |
- Mean unenriched purity measured across three runs
Table 11 shows the FISH analysis results for the selected enriched samples from each panel member and run. For low initial CD138+ frequency panel members, the number of abnormal nuclei per slide ranged from 85.5% - 94.0% and replicates from all panel members and runs resulted in abnormal FISH signal patterns. For medium initial CD138+ frequency members, the number of abnormal nuclei per slide ranged from 93% -99.5%, and replicates from all panel members and runs resulted in abnormal FISH signal patterns. Therefore, the acceptance criteria for within laboratory precision by FISH analysis were met.
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Table 11. FISH Analysis Results for Enriched Samples bv Run for Low and Medium Initial CD138 Frequency Panel Members
| Initial CD138FrequencyCategory | PanelMember | Run | Replicate(a/b/c/d) | CD 138 Purity (% CD138+) | FISH Results | ||
|---|---|---|---|---|---|---|---|
| Unenriched | Enriched | # AbnormalNuclei | Normal/Abnormal | ||||
| Low | B1 | 1 | b | 1.51 | 87.48 | 92.5 | Abnormal |
| 2 | d | 83.42 | 94 | Abnormal | |||
| 3 | a | 84.41 | 93 | Abnormal | |||
| B4 | 1 | d | 1.32 | 85.65 | 90 | Abnormal | |
| 2 | c | 87.93 | 89.5 | Abnormal | |||
| 3 | a | 87.44 | 94 | Abnormal | |||
| B5 | 1 | b | 1.37 | 80.99 | 85.5 | Abnormal | |
| 2 | c | 83.77 | 86 | Abnormal | |||
| 3 | d | 82.12 | 89.5 | Abnormal | |||
| Medium | B2 | 1 | a | 10.84 | 95.62 | 99.5 | Abnormal |
| 2 | b | 93.57 | 96.5 | ||||
| 3 | d | 92.75 | 97 | ||||
| B3 | 1 | b | 9.84 | 95.43 | 94.5 | Abnormal | |
| 2 | d | 94.81 | 95.5 | Abnormal | |||
| 3 | c | 96.14 | 94 | Abnormal | |||
| B6 | 1 | a | 10.17 | 91.1 | 93 | Abnormal | |
| 2 | a | 93.69 | 93.5 | Abnormal | |||
| 3 | b | 94.21 | 97.5 | Abnormal |
The CD138 EasySep Precision study showed that the EasySep Human Bone Marrow CD138 Positive Selection Kit produced consistent enrichment within a run and across three runs when starting with a specimen of a given volume and initial CD138+ cell frequency. For low initial CD138+ frequency panel members (n=3) flow cytometry analysis, the repeatability precision estimate (n=4 per run) for enriched CD138+ purity CV ranged from 0.81% to 1.56% and the within-laboratory precision estimate (n=12 across 3 runs) for enriched CD138+ purity CV ranged from 0.84% to 1.77%. For medium initial CD138+ frequency panel members (n=3) flow cytometry analysis, the repeatability precision estimate (n= 4 per run) for enriched CD138+ purity CV ranged from 0.17% to 1.41% and the within-laboratory precision estimate (n=12 across 3 runs) for enriched CD138+ purity CV ranged from 0.65% to 0.96%. All enriched samples sent for FISH analysis (n=3 per panel member, one replicate per run) gave abnormal FISH patterns, demonstrating within-laboratory precision in downstream FISH assays. Overall, the precision of the EasySep Human Bone Marrow CD138 Positive Selection Kit met the acceptance criteria set for the precision study.
-
- Analytical Specificity/Interference:
Three (3) fresh clinical multiple myeloma bone marrow specimens were tested to determine if excess heparin affected device performance. One specimen was processed at ~20 hours post collection, one specimen was processed at ~24 hours post collection, and the third specimen was processed ~22 hours post collection. Each specimen was split into two fractions. One fraction was treated with phosphate buffered saline (PBS, the control), while the other was spiked with 3x excess of sodium heparin. Single replicates of each fraction were then washed and enriched using the EasySep Human Bone Marrow CD138 Positive Selection Kit. CD138 purity was determined by flow cytometry and then
- Analytical Specificity/Interference:
{10}------------------------------------------------
compared to determine whether excess heparin caused interference with CD138 enrichment and purity. The enriched CD138+ purity from the test samples with excess sodium heparin should be > 90% of the enriched CD138+ purity from the control samples without excess heparin.
Table 12 shows CD138 purity before plasma cell enrichment and after enrichment using the EasySep Human Bone Marrow CD138 Positive Selection Kit for each specimen. For specimen #1, the PBS control sample was enriched to 64.7% and the excess sodium heparin-containing sample was enriched to 58.7%. which is 91.0% of the control. For specimen #2, the PBS control sample was enriched to 32.4% and the excess sodium heparin containing sample was enriched to 33.4%, which is 103% of the PBS control. For specimen #3, the PBS control sample was enriched to 81.7% and the excess sodium heparin-containing sample was enriched to 80.1%. which is 98.0% of the control. All three specimens passed the set acceptance criteria for the study, which was that enriched CD138+ purity from the test samples with excess sodium heparin should be > 90% of the enriched CD138+ purity from the control samples without excess heparin.
| Specimen# | Initial CD138Frequency(%) | After Enrichment | CD138 Purity:Heparin/ PBSControl | Meets AcceptanceCriteria?(Pass/Fail) | |
|---|---|---|---|---|---|
| PBS Control(%) | + Heparin(%) | ||||
| 1 | 5.0 | 64.7 | 58.7 | 91.0 | PASS |
| 2 | 0.4 | 32.4 | 33.4 | 103.0 | PASS |
| 3 | 31.3 | 81.7 | 80.1 | 98.0 | PASS |
Table 12. Effect of Excess Heparin on CD138 Purity
A second study evaluated a total of 3 clinical specimens prepared by adding MM bone marrow mononuclear cells into healthy donor bone marrow at <0.2% initial CD138 frequency. Three (3) replicates were tested per specimen per condition (i.e., with/without excess sodium heparin). The enriched purities of heparin spiked specimens were not statistically different from that of the control specimens (one tailed t-test, p=0.2740).
Table 13 shows CD138 purity before plasma cell enrichment and after enrichment using the EasySep Human Bone Marrow CD138 Positive Selection Kit for each specimen. The mean enriched purity for Clinical Specimen 1 was 84.45% for the Control Specimen and 83.97% for the Heparin-spiked Specimen, which was 99% of Control. The mean enriched purity for Clinical Specimen 2 was 96.37% for the Control Specimen and 96.25% for the Heparin-spiked Specimen, which was 100% of Control. The mean enriched purity for Clinical Specimen 3 was 88.90% for the Control Specimen and 89.08% for the Heparinspiked Specimen, which was 100% of Control. The enriched purities of heparin spiked specimens were not statistically different from the enriched purities of control specimens (one tailed t-test, p=0.2740, N=3). All three clinical specimens passed the acceptance criteria that enriched CD138+ purity from the test samples with excess sodium heparin should be > 90% of the enriched CD138+ purity from the control samples without excess heparin.
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| ClinicalSpecimen | TargetInitial %CD138 | Replicate | Enriched Purity(% CD138+) | Mean EnrichedPurity (% CD138+) | Heparin/Control (%) | AcceptanceCriteria(Pass/Fail) | ||
|---|---|---|---|---|---|---|---|---|
| 1 | 0.14 | 1 | 83.52 | 81.69 | 84.45 | 83.97 | 99 | Pass |
| 1 | 0.14 | 2 | 85.26 | 83.23 | ||||
| 1 | 0.14 | 3 | 84.57 | 86.99 | ||||
| 2 | 0.14 | 1 | 96.45 | 96.26 | 96.37 | 96.25 | 100 | Pass |
| 2 | 0.14 | 2 | 96.74 | 96.21 | ||||
| 2 | 0.14 | 3 | 95.92 | 96.27 | ||||
| 3 | 0.19 | 1 | 89 | 90.05 | 88.90 | 89.08 | 100 | Pass |
| 3 | 0.19 | 2 | 88.79 | 89.24 | ||||
| 3 | 0.19 | 3 | NA | 87.96 |
Table 13. Summary of CD138 Purity from Low Frequency Clinical Specimens
Results show the enriched purities from the excess anticoagulant containing samples were not statistically different from those of the control samples. Therefore, the study results demonstrate that there is no interference identified from the excess anticoagulant used when enriching samples with the EasySep Human Bone Marrow CD138 Positive Selection Kit.
4. Stability
Specimen Stability
The CD138 Expression Stability Study aimed to process ~six MM patient BMA at baseline (<24 hours) and at either 44 - 54 hours or ~73 hours post-draw. One kit lot of the EasySep Human Bone Marrow CD138 Positive Selection kit was used for each time point. At each time point, flow cytometry was performed for both unenriched and enriched portions of each sample, and FISH was only performed on the enriched portion of each sample. For FISH analysis, all enriched samples should have conclusive FISH results. Each of the five FISH probes should result in the same FISH disposition (i.e., normal or abnormal).
CD138 purity before and after enrichment and FISH results using five common FISH probes for each time point are shown in Table 14. Specimen #1 had an initial CD138 frequency of 10.6%, which dropped to 3.1% at 73 hours post-collection. Following enrichment, plasma cells were enriched to 89.9% at baseline, and decreased to 62.4% at 73 hours. FISH results for the three probes analyzed did not change across the time points. Specimen #2 had a very low initial CD138 frequency of 0.5%, which decreased below the limit of quantitation (LLoQ) for the flow assay at 44 - 54 hours and further decreased to below the limit of detection (LLoD) for the flow assay at 73 hours postcollection. As such, data are reported as <LLoQ and <LLoD. FISH results were consistent with baseline and 44 - 54 hours, but were more variable at ~73 hours, as there were insufficient cells to obtain conclusive FISH results for two of the probes. The initial CD138 frequency could not be determined at this time point and could therefore be below the LoD of 0.05% that has been determined for the EasySep Human Bone Marrow CD138 Positive Selection Kit. Specimen #3 also had a very low initial CD138 frequency of 0.4%. At ~73 hours post-collection, purity was again below the LLoD for the flow assay and could not be determined. FISH disposition for the three probes analyzed was consistent across the time points.
{12}------------------------------------------------
| Bone MarrowSpecimen | FISH Probe | Baseline (<24 h) | 44 - 54 h | ~73 h |
|---|---|---|---|---|
| 1 | Initial CD138 Frequency | 10.6% | 9.1% | 3.1% |
| Enriched CD138 Purity | 89.9 | 85.1 | 62.4 | |
| Chr. 13 (D13S319) | Abnormal | Abnormal | Abnormal | |
| CCND1/IGH XT | Normal | Normal | Normal | |
| TP53 | Normal | Normal | Normal | |
| 2 | Initial CD138 Frequency | 0.5% | <LLOQ**(not quantifiable) | <LLOD**(not detected) |
| Enriched CD138 Purity | 36.7 | 30.8 | 28.9 | |
| Chr. 13 (D13S319) | Abnormal | Abnormal | QNS | |
| CCND1/IGH XT | Normal | Normal | Normal | |
| TP53 | Normal | Normal | QNS | |
| 3 | Initial CD138 Frequency | 0.4% | Not tested | <LLOD**(not detected) |
| Enriched CD138 Purity | 32.4 | Not tested | 23.2& | |
| Chr. 13 (D13S319) | Normal | - | Normal | |
| CCND1/IGH XT | Normal | - | Normal | |
| TP53 | Normal | - | Normal |
Table 14. Stability of CD138 Expressing Multiple Myeloma Bone Marrow Aspirates
*QNS - Quantity of cells not sufficient for analysis
** CD138 purity values are below the putative LLoO of 0.14% or LLoD of 0.14% for the flow cytometry assay performed and could not be determined.
& Note: This sample was processed at 69 hours post-collection instead of 73 hours.
Overall, FISH disposition was consistent between baseline and ~73 hours for all three specimens when initial CD138 frequencies were above the putative LLoD for the flow assay (i.e., 0.14%). Although CD138 specimens could be enriched at ~73 hours, FISH signals were not reliable when specimens with low initial CD138 frequencies were too low to be determined to be determined by flow cytometry.
Kit Stability
Shelf-Life and In-Use
This study was conducted to establish that the EasySep Human Bone Marrow CD138 Positive Selection Kit is able to maintain its performance characteristics over its defined shelf-life when stored at 5℃ ± 3℃ using three kit lots. The test samples were stored unopened at 5°C ± 3°C. Contrived samples were tested at or to be test at different time points (0, 3, 7, 10, 13, 19 and 25 months). Three kit lots were tested for shelf-life and 1 lot was tested for in-use stability. The in-use testing involved the mixing and removal of 25 uL from the cocktail and particle vials for 20 times over a 7-month duration before the samples were tested. Kits had to demonstrate ability to enrich CD138+ cells to a purity of >95% and >70% recovery.
Stability testing of three lots of EasySep Human Bone Marrow CD138 Positive Selection Kit has met all acceptance criteria at the long-term and in-use storage condition of 5℃ ± 3°C for up to 7 months and at the accelerated storage condition of 25°C ± 2°C/ 60% ± 5% RH for up to 7 days. The data therefore support a 6-month shelf life for EasySep Human Bone Marrow CD138 Positive Selection Kit stored at 5°C ± 3°C. The data also
{13}------------------------------------------------
supports the stability of the products for up to 7 days at 25℃ ± 2℃/ 60% ± 5% relative humidity.
Shipping
This study summarizes data collected for up to 7 months at the long-term storage condition of 5°C ± 3°C from the shipping stability study conducted on one batch of EasySep Human Bone Marrow CD138 Positive Selection Kit. Shipping stability kits packaged in their final packaging configurations were subjected to summer and winter temperature profiles for 48 hours and tested for enrichment performance and FISH and monitored with data loggers. For the 48-hour Summer Profile Testing, the test article was exposed to temperature test cycles with a recorded maximum temperature of 30.5 °C and minimum of 23.8 ℃. For the 48-hour winter profile testing, the maximum recorded temperature to which the test article was exposed to is 15.1℃ while the minimum recorded temperature is 3.6 ℃. Kits had to demonstrate that CD138+ cell purity had to be >95% with at least 70% of cells recovered. All acceptance criteria were met at the longterm storage condition of 5°C ± 3°C for up to 7 months.
The stability data demonstrated that transport of the EasySep Human Bone Marrow CD138 Positive Selection Kit using the qualified 48-hour shipping configuration does not affect product performance characteristics. The data support a 6-month shelf-life following transport stress. The shipping stability study will continue for up to 25 months.
5. Detection Limit:
a) Limit of Detection with Contrived Samples
The study aimed to determine the lowest initial CD138+ frequency that gives consistent abnormal FISH results in a contrived specimen after CD138+ cell enrichment using the EasySep Human Bone Marrow CD138 Positive Selection Kit. On six non-consecutive days, a whole blood (WB) or BM sample from a healthy donor was spiked with the CD138+ cell line SK-MM-2 at 20% (acceptable range 26% -14% by flow cytometry) CD138+ initial frequency and then serially diluted by a factor of two with WB or BM to achieve ~20%, 10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.08%, and 0% (i.e., WB or BM not spiked with SK-MM-2 cells). Each sample was enriched in duplicate using the EasySep Human Bone Marrow CD138 Positive Selection Kit and all unenriched and enriched specimens were assessed by flow cytometry.
Table 15 shows the results from the EasySep CD138 Limit of Detection Study including the unenriched and enriched CD138+ cell purity measured by flow cytometry and the t(11:14) disposition analyzed by FISH. All contrived specimens spiked with SK-MM-2 cells, targeting as low as 0.08% initial CD138+ frequency, resulted in abnormal t(11;14) disposition in FISH analysis.
The enriched CD138+ purity measured by flow cytometry positively correlated with the percentage of abnormal nuclei observed in downstream FISH analysis. The average enriched CD138+ purity that resulted in abnormal t(11;14) disposition in FISH was 33% CD138+, enriching from specimens with an average unenriched purity of 0.07% CD138+. For 0.00% initial target CD138+ frequency specimens, four out of the six donors resulted in normal t(11:14) disposition in FISH analysis, however one BM and one WB donor only had one replicate deemed normal and the other replicate inconclusive
{14}------------------------------------------------
due to insufficient number of cells recovered. Since the lowest targeted initial CD138+ frequency specimen achieved abnormal t(11;14) disposition by FISH, the limit of detection (i.e., the lowest initial CD138+ frequency in a contrived specimen that gave a consistent abnormal t(11;14) disposition in FISH after CD138+ enrichment) was determined to be in the range of 0.05 - 0.15% CD138+ measured by flow cytometry. The lower end of the range was below the limit of detection of the flow cytometer. Therefore, the limit of detection was set at the limit of detection of the flow cytometer, which was 0.05% CD138+.
| TargetCD138+InitialFrequency | Flow Cytometry Analysis | FISH Analysis | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Unenriched CD138+ Purity (%) | Enriched CD138+ Purity (%) | %Abnormal Nuclei and t(11;14)Disposition | ||||||||||||
| Mean | Min | Max | N | Mean | Min | Max | N | Mean | Min | Max | Normal/Abnormal | N | ||
| 0.00% | NA | ND | <LoQ | ND: 5<LoQ: 1 | 13.33 | <LoQ | 32.48 | 12 | 0.0 | 0.0 | 0.0 | NormalInconclusive | N=10N=2 | |
| 0.08% | 0.09* | 0.05* | 0.15* | 6 | 33.03 | 6.31 | 68.07 | 12 | 51.7 | 30.5 | 73.0 | Abnormal | 12 | |
| 0.16% | 0.18* | 0.10* | 0.30* | 6 | 42.57 | 11.52 | 76.2 | 12 | 68.0 | 40.0 | 82.0 | Abnormal | 12 | |
| 0.31% | 0.32* | 0.21* | 0.40* | 6 | 55.42 | 20.73 | 83.82 | 12 | 83.4 | 73.5 | 90.5 | Abnormal | 12 | |
| 0.63% | 0.69 | 0.41 | 0.94 | 6 | 67.2 | 30.97 | 90.12 | 12 | 87.6 | 80.0 | 92.5 | Abnormal | 12 | |
| 1.25% | 1.38 | 0.87 | 1.86 | 6 | 76.55 | 51.55 | 90.43 | 12 | 93.1 | 85.5 | 97.0 | Abnormal | 12 | |
| 2.50% | 2.17 | 1.41 | 2.84 | 6 | 87.07 | 76.24 | 93.86 | 12 | 95.8 | 89.5 | 99.0 | Abnormal | 12 | |
| 5% | 5.28 | 3.37 | 7.25 | 6 | 91.71 | 88.26 | 95.01 | 12 | 97.0 | 93.5 | 100.0 | Abnormal | 12 | |
| 10% | 10.71 | 8.16 | 13.21 | 6 | 94.39 | 91.2 | 96.91 | 12 | 98.0 | 96.5 | 99.5 | Abnormal | 12 | |
| 20% | 20.01 | 14.73 | 25.06 | 6 | 95.01 | 92.93 | 98.2 | 12 | 98.1 | 96.5 | 100.0 | Abnormal | 12 |
Table 15. EasySep CD138 Limit of Detection Flow Cytometry and FISH Analysis Result Summary
Table 16 shows the unenriched and enriched purities for BM and WB contrived specimens for each target initial frequency. The healthy donor BM only specimens (0% initial CD138+ frequency) had enriched CD138+ purities in the range of 16.7% - 32.5%.
| Table 16. Unenriched and Enriched Purities for BM and WB Contrived Specimens for Each | |||
|---|---|---|---|
| Target Initial Frequency |
| TargetInitialCD138+Frequency | Sample Type | |||||||
|---|---|---|---|---|---|---|---|---|
| BM | WB | |||||||
| Unenriched Purity(% CD138+)Mean | Enriched Purity(% CD138+)Mean | Enriched Purity(% CD138+)Min | Enriched Purity(% CD138+)Max | Unenriched Purity(% CD138+)Mean | Enriched Purity(% CD138+)Mean | Enriched Purity(% CD138+)Min | Enriched Purity(% CD138+)Max | |
| 0 | 0.04 | 26.21 | 16.66 | 32.48 | 0.00 | 0.46 | 0.10 | 0.98 |
| 0.08 | 0.07 | 54.67 | 39.73 | 68.07 | 0.07 | 11.40 | 6.31 | 16.10 |
| 0.16 | 0.18 | 65.4 | 54.28 | 76.2 | 0.12 | 19.75 | 11.52 | 33.01 |
| 0.31 | 0.31 | 75.61 | 69.86 | 83.82 | 0.21 | 35.23 | 20.73 | 55.57 |
| 0.63 | 0.71 | 82.35 | 74.86 | 90.12 | 0.67 | 52.05 | 30.97 | 72.51 |
| 1.25 | 1.29 | 85.35 | 78.84 | 90.43 | 1.47 | 67.74 | 51.55 | 81.51 |
| 2.5 | 2.33 | 89.78 | 83.92 | 93.86 | 2.01 | 84.37 | 76.24 | 91.95 |
| 5 | 5.82 | 91.87 | 89.21 | 95.01 | 4.74 | 91.56 | 88.26 | 93.93 |
| 10 | 10.41 | 93.75 | 91.20 | 95.94 | 11.01 | 95.03 | 92.83 | 96.91 |
| 20 | 19.84 | 94.34 | 92.93 | 96.87 | 20.18 | 95.68 | 94.06 | 98.2 |
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b) Limit of Detection with Clinical Multiple Myeloma Bone Marrow Specimens
Five (5) low CD138 frequency specimens with pre-enrichment frequencies of 0.2%, 0.5%, 0.7%, 1.5%, and 1.7% CD138+ (Table 17). All five specimens were successfully enriched to purity ≥ 18.5% CD138+ with fold enrichment > 29.6-fold. All five specimens had an increase in the percentage of abnormal nuclei in FISH analysis for at least one FISH probe. Three of the five specimens only had percentages of abnormal nuclei above FISH cutoff after plasma cell enrichments (highlighted in bold). The data confirm that clinical MM BM specimens with initial CD138+ frequency close to the limit of detection set in this study were able to have plasma cells successfully enriched and gave interpretable FISH results.
Table 17. CD138 Purity and Percentage of Abnormal Nuclei Detected by FISH Probes Pre- and Post-EasySep CD138 Enrichment for Low CD138 Initial Frequency Multiple Myeloma Bone Marrow Aspirates
| MMPatientSample | CD138Freq* | Pre- EnrCD138Purity | EnrCD138Purity | FoldEnr& | % Abnormal Nuclei | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Probe(D5S23)Cutoff =1% | Probe(D15Z4)Cutoff =1% | Probe(MYC)Cutoff =3% | Probe(D13S319,13qter) Cutoff= 14% | Probe(IGH/CCND1 XT)Cutoff=3-15% | Probe (TP53,D17Z1) Cutoff= 9% | |||||||||||
| Pre-Enr | Enr | Pre-Enr | Enr | Pre-Enr | Enr | Pre-Enr | Enr | Pre- Enr | Enr | Pre-Enr | Enr | |||||
| BM 118 | low | 0.7 | 64.5 | 91.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 85.5 | 0 | 0 |
| BM 121 | low | 0.2 | 18.5 | 91.5 | 1.5 | 48 | 0 | 0 | 0 | 0 | 0 | 50 | 0 | 0 | 0 | 0 |
| BM 122 | low | 1.7 | 52.0 | 29.6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 80 | 0 | 0 | 0 | 0 |
| 5 | low | 1.5 | 80.6 | 52.7 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 49 | 0 | 77 | 0 | 0 |
| 8 | low | 0.5 | 32.8 | 64.6 | 5 | 53 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Enr = enrichment
*low = < 3% CD138 start frequency
& Fold enrichment is calculated using the following formula: [Enriched %CD138 Purity / Pre-enriched %CD138 Purity] - 1
The limit of detection study showed that the EasySep Human Bone Marrow CD138 Positive Selection Kit was able to consistently enrich from contrived specimens with initial CD138+ frequency of 0.02% - 25.06% and achieved consistent abnormal FISH patterns even in the lowest initial target CD138+ frequency specimens spiked with the SK-MM-2 cell line. Therefore, the limit of detection for the EasySep Human Bone Marrow CD138 Positive Selection Kit was set to 0.05% of initial CD138+ frequency. which is the limit of detection of the flow cytometer used.
c) Verification of Limit of Detection with Clinical Multiple Myeloma Bone Marrow Specimens
The goal of this study was to verify the LoD at 0.05% initial CD138 frequency, determined in a previous LoD study on contrived specimens in one clinical specimen using two kit lots, having 3 replicates per kit lot. CD138+ cell enrichment was performed on a clinical specimen produced by spiking a multiple myeloma patient BM aspirate into a healthy donor BM aspirate at 0.05% initial CD138 frequency. The clinical specimen was washed and split into 6 fractions for EasySep CD138 cell enrichments, using two kit lots with three replicates per kit lot. Enriched samples were split into two fractions: one fraction was used for plasma cell purity assessment by flow cytometry and the second fraction was used for FISH.
{16}------------------------------------------------
Table 18 shows plasma cell purity before and after enrichment using two kit lots of the EasySep Human Bone Marrow CD138 Positive Selection Kit with three replicates per kit lot. The specimen was enriched from 0.05% to a range of 4.1% to 7.4%. Overall enriched purity CV was 19.7%, passing the acceptance criteria of overall CV ≤ 35%. All enriched samples had normal FISH signal patterns for Chromosome 5 and 15 polysomy (D5S23. D15Z4), MYC breakapart, and the CCND1/IGH XT probes; and were abnormal for Chromosome 13q deletion (D13S319) and TP53. The enriched samples' FISH disposition was the same as the undiluted clinical specimen's FISH disposition, with 100% of the replicates being abnormal for Chromosome 13g deletion (D13S319) and TP53.
| Sample | UnenrichedPlasma CellPurity (%per 105events) | Enriched Plasma Cell Purity byFlow Cytometry | Genomic Abnormality by FISH | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| % PlasmaCell per105events | Mean | SD | %CV | D5S23 | D15Z4 | MYC | D13S319,13qter | TP53,D17Z1 | IGH/CCND1(LDT) | ||
| UndilutedMM BMA | 12.9 | - | - | - | - | Normal | Normal | Normal | Abnormal | Abnormal | Normal |
| Lot 1 - A | 0.05 | 4.1 | 6.2 | 1.2 | 19.7 | Normal | Normal | Normal | Abnormal | Abnormal | Normal |
| Lot 1 - B | 7.4 | Normal | Normal | Normal | Abnormal | Abnormal | Normal | ||||
| Lot 1 - C | 7.0 | Normal | Normal | Normal | Abnormal | Abnormal | Normal | ||||
| Lot 2 - A | 5.8 | Normal | Normal | Normal | Abnormal | Abnormal | Normal | ||||
| Lot 2 - B | 7.0 | Normal | Normal | Normal | Abnormal | Abnormal | Normal | ||||
| Lot 2 - C | 5.8 | Normal | Normal | Normal | Abnormal | Abnormal | Normal |
| Table 18. Summary of Limit of Detection Results for Clinical Specimens | |||||||
|---|---|---|---|---|---|---|---|
| -- | -- | -- | -- | ------------------------------------------------------------------------ | -- | -- | -- |
B Clinical Studies:
1. Clinical Study
This study summarizes CD138 purity data and FISH data generated on bone marrow aspirates (BMA) from 33 multiple myeloma patients at various stages of disease collected in two studies. In both studies, one portion of the sample was retained as the pre-enriched sample, and the other portion was split again and enriched with the EasySep Human Bone Marrow CD138 Positive Selection Kit. CD138 purity was assessed by flow cytometry on both the pre-enriched and enriched samples. Each sample (pre-enriched and enriched) was then hybridized with five common FISH probe kits that included the CCND1/IGH XT Probe Kit to detect the t(11:14) translocation, as well as probes to detect chromosome 5 (D5S23) and chromosome 15 polysomy (D15Z4) (single probe kit), MYC breakapart, 13q deletion (D13S319), and TP53 deletion (D17Z1).
Specimen slides were enumerated by a minimum of two readers; 50 nuclei per reader were analyzed, for a total of 100 nuclei. If counts for a reader were below or equal to the cutoff corresponding to each probe, then the result from the reader was determined to be normal and the percentage of abnormal nuclei would be indicated as '0' (Table 19).
Thirty-three BMAs from patients at various stages of disease were enriched using the EasySep Human Bone Marrow CD138 Positive Selection Kit. CD138 purity was determined by flow cytometry and the percentage of abnormal nuclei detected for five common FISH probes for all pre-enriched and enriched specimens. CD138 purity data
{17}------------------------------------------------
pre and post EasySep enrichment was collected by flow cytometry for 30 of the 33 patients. CD138 purities prior to enrichment ranged from 0.2% to 82.7% (average 12.9%); five specimens had CD138+ plasma cell purities of <3% prior to enrichment, 16 had purities between 3% and 15%, and nine had purities >15%. Purities after EasySep enrichment ranged from 18.5% to 98.6% (average 79.6%).
| Table 19. CD138 Purity and Percentage of Abnormal Nuclei Detected or Five FISH Probes Pre- | |
|---|---|
| and Post-EasySep CD138 Enrichment for 33 Multiple Myeloma Bone Marrow Aspirates |
| MMPatientSample | CD138Frequency* | Pre-EnrCD138Purity | EnrichedCD138Purity | FoldEnrich-ment& | Probe(D5S23) | Probe(D15Z4) | Probe(MYC) | Probe(D13S319,13qter) | Probe(IGH/CCND1XT) | Probe(TP53,D17Z1) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pre-Enr | Enr | Pre-Enr | Enr | Pre-Enr | Enr | Pre-Enr | Enr | Pre-Enr | Enr | Pre-Enr | Enr | |||||
| 1 | medium | 5.0 | 57.3 | 10.5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 51 | 13 | 53 | 0 | 0 |
| 2 | high | 15.5 | 91.4 | 4.9 | 35.5 | 87 | 21.5 | 63 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 3 | high | 15.6 | 98.2 | 5.3 | 0 | 0 | 0 | 0 | 0 | 17 | 19 | 88 | 0 | 0 | 0 | 0 |
| 4 | high | 82.7 | 77.7 | -0.1 | 38 | 45 | 34.5 | 30 | 69 | 91 | 50.5 | 74 | 0 | 0 | 54 | 70 |
| 5 | medium | 4.3 | 57.0 | 12.3 | 25.5 | 74 | 28.5 | 67 | 0 | 0 | 0 | 0 | 0 | 8.5 | 0 | 0 |
| 6 | medium | 6.5 | 98.6 | 14.2 | 0 | 0 | 19 | 44 | 25.5 | 46 | 14.5 | 73 | 32.5 | 92 | 26.5 | 77 |
| 7 | medium | 10.0 | 94.4 | 8.4 | 20 | 69 | 17.5 | 71 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 8 | high | 36.2 | 93.7 | 1.6 | 45 | 88 | 0 | 0 | 39 | 53 | 0 | 0 | 54 | 94 | 0 | 0 |
| 9 | medium | 13.8 | 92.0 | 5.7 | 0 | 0 | 0 | 0 | 29.5 | 89 | 24.5 | 89 | 0 | 0 | 0 | 0 |
| 10 | high | 31.4 | 85.7 | 1.7 | 0 | 0 | 9 | 75 | 0 | 0 | 0 | 90 | 18 | 95.5 | 0 | 0 |
| 11 | medium | 7.9 | 85.6 | 9.8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 95 | 9.5 | 91.5 | 0 | 0 |
| 12 | high | 16.2 | 91.0 | 4.6 | 29.5 | 85 | 16 | 51 | 0 | 0 | 41 | 93 | 21.5 | 86.5 | 0 | 0 |
| 13 | medium | 3.7 | 88.9 | 23.0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 14 | medium | 12.4 | 75.3 | 5.1 | 14.5 | 47 | 0 | 0 | 23.5 | 88 | 24 | 97 | 0 | 0 | 9.5 | 43 |
| 15 | medium | 7.7 | 81.7 | 9.6 | 0 | 0 | 17 | 53 | 0 | 6 | 20.5 | 93 | 0 | 8.5 | 0 | 0 |
| 16 | high | 35.0 | 95 | 1.7 | 0 | 0 | 0 | 0 | 0 | 0 | 40 | 88 | 0 | 0 | 46.5 | 81 |
| 17 | medium | 4.6 | 82.9 | 17.0 | 0 | 0 | 0 | 0 | 0 | 0 | 18 | 77 | 8 | 82 | 0 | 22 |
| 18 | low | 0.7 | 64.5 | 91.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 85.5 | 0 | 0 |
| 19 | medium | 4.9 | 88.3 | 17.0 | 10.5 | 75 | 7 | 27 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 20 | high | 27.7 | 96.8 | 2.5 | 77 | 91 | 68 | 81 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 21 | low | 0.2 | 18.5 | 91.5 | 1.5 | 48 | 0 | 0 | 0 | 0 | 0 | 50 | 0 | 0 | 0 | 0 |
| 22 | low | 1.7 | 52.0 | 29.6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 80 | 0 | 0 | 0 | 0 |
| 23 | high | 17.2 | NC | n/a | 15 | 95 | 16 | 93 | 0 | 0 | 0 | 0 | 29 | 92 | 0 | 0 |
| 24 | medium | 5.7 | NC | n/a | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 90 | 0 | 0 | 0 | 0 |
| 25 | low | 2.3 | NC | n/a | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 7 | 64 | 0 | 0 |
| 26 | medium | 10.6 | 96.3 | 8.1 | 8.5 | 68 | 9 | 73 | 7.5 | 37 | 0 | 0 | 0 | 0 | 0 | 0 |
| 27 | low | 1.5 | 80.6 | 52.7 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 49 | 0 | 77 | 0 | 0 |
| 28 | high | 20 | 92.1 | 3.6 | 0 | 0 | 4 | 61 | 0 | 0 | 40 | 91 | 0 | 0 | 0 | 0 |
| 29 | medium | 9.7 | 90.6 | 8.3 | 21.5 | 75 | 13.5 | 55 | 0 | 8 | 18.5 | 84 | 0 | 0 | 0 | 0 |
| 30 | low | 0.5 | 32.8 | 64.6 | 5 | 53 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 31 | medium | 5 | 84.6 | 15.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 83 | 0 | 0 |
| 32 | medium | 5.8 | 87.5 | 14.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 80 | 0 | 0 | 0 | 0 |
| 33 | medium | 4.3 | 56.2 | 12.1 | 28 | 91 | 28 | 87 | 18 | 72 | 0 | 96 | 0 | 0 | 0 | 0 |
{18}------------------------------------------------
Enr = enriched
*low = < 3% CD138 start frequency; medium = 3-15% CD138 start frequency; high =>15% CD138 start frequency & Fold enrichment is calculated using the following formula: [Enriched %CD138 Purity / Pre-enriched %CD138 Purity] - 1 NC - not collected
CD138 purity increased from 12.9% ± 15.9% in the pre-enriched sample to 79.6% ± 19.9% following enrichment. Fold enrichment was > 1 for all specimens with CD138 frequencies <40% prior to enrichment. In 14 patients, abnormal nuclei were only detected after the sample had been enriched using the CD138 kit (bold). For all patients, the percentage of abnormal nuclei detected was higher in the enriched sample compared to the unenriched sample indicating an increase in the sensitivity of the FISH assay. This data demonstrates that the EasySep Human Bone Marrow CD138 Positive Selection kit can be used to enrich CD138+ plasma cells from multiple myeloma patients for use in detection of multiple FISH probes.
VII Proposed Labeling:
The labeling supports the decision to grant the De Novo request for this device.
| Risks to Health | Mitigation Measures |
|---|---|
| Failure to perform as expected due to errors inenrichment, contributing to false positive orfalse negative results, or failure to produceresults in downstream assays | Use of certain specimen collection devices.Certain design verification and validation,including certain studies and risk mitigationanalysis.Certain labeling information, includinglimitations, device descriptions, methodologyand protocols, and performance information. |
| Incorrect interpretation of enrichment resultsby the lab: | Certain labeling information, includinglimitations, device descriptions, methodologyand protocols, and performance information.Certain design verification and validation,including certain studies and risk mitigationanalysis. |
VIII Identified Risks and Mitigations:
IX Benefit/Risk Assessment:
A Summary of the Assessment of Benefit:
The EasySep Human Bone Marrow CD138 Positive Selection Kit has probable benefit given the totality of data provided, which indicate that use of this device provides enriched and purified CD138+ plasma cells (both benign and malignant), for downstream applications like molecular assays and flow cytometry. In addition, in the clinical studies, the sponsor was successfully able to enrich a range of bone marrow CD138+ cells (low, intermediate, and high), enriching the CD138+ cells with their device, and showed that they were able to successfully perform FISH with these viable, enriched cells. A key benefit of this device is that it increases the concentration of the cells of interest in samples with low concentrations of specific hematopoietic cells, such as those in patients undergoing treatment or relapsed refractory patients, which will help decrease the false negative rate for any test. Patients and
{19}------------------------------------------------
physicians will have a better idea of the molecular genetics of their disorder, because this selection kit allows for enrichment of CD138+ plasma cells, such that cytogenetic and molecular analysis can be performed with enriched cells. This may lead to better clinical . management for patients with multiple myeloma or other hematopoietic malignancies.
B Summary of the Assessment of Risk:
The probable risks of this device include the malfunction of CD138+ plasma cell purification, which may impact downstream assays, are: 1) failure to provide a result due to errors in enrichment, contributing to false positive or false negative results or failure to produce results in assays and 2) incorrect interpretation of enrichment results by the lab. Poor enrichment of plasma cells may lead to false negative results, or no results reported. There is also a degree of probable risk regarding false positives; however, the risk of false positivity is thought to be low, due to technical reasons.
In the validation of this device, not all downstream assays were tested. The user is responsible for the adequate validation of downstream assay. The device has limitations where very low levels of malignant plasma cells may not be able to be sufficiently enriched for downstream applications.
The risks outlined above, are mitigated by use of specimen collection devices. substantive design verification and validation studies (including supportive analytical and clinical studies), labeling information, including limitations, device descriptions, methodology and protocols, and performance information. In addition, a risk mitigation analysis was performed, indicating that the major sources of risk were adequately mitigated.
C Patient Perspectives:
This submission did not include specific information on patient perspectives for this device.
D Summary of the Assessment of Benefit-Risk:
The probable benefit of the device was demonstrated by a high percentage of cell viability after enrichment and studies that showed that the enriched cells vielded interpretable results from downstream molecular analysis. The limitations of the enrichment and potential variability in enrichment were also shown. In addition, the performance of the device was supported by studies such as the limit of detection and precision, which inform users of the limitations of this device and serve to mitigate risks of this device. Therefore, while general controls are not sufficient to address the risks of this device, when taking into account the special controls, the probable benefits of this device are deemed to outweigh the probable risks of this device.
X Conclusion:
The De Novo request is granted, and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:
Product Code(s): QYO Device Type: Hematopoietic cell enrichment kit Class: Class II Regulation: 21 CFR 866.6120
N/A