Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a kit for immunomagnetic cell enrichment, which is a physical separation method. There is no mention of AI, ML, image processing, or any computational analysis that would suggest the use of these technologies. The performance studies focus on the biological and physical aspects of the enrichment process and downstream assays like flow cytometry and FISH.

Therapeutic

No.
This device is an in vitro diagnostic device used to enrich cells for downstream laboratory assays, not to directly treat a patient.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device "is an in vitro diagnostic device." It is used to enrich cells for use in "validated downstream assays," which are used to diagnose multiple myeloma.

SaMD (Software as a Medical Device)

No

The device is a kit containing reagents for immunomagnetic positive selection, which are physical components, not software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "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."

This statement directly identifies the device as an in vitro diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications 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.

Product codes

QYO

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.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

bone marrow

Indicated Patient Age Range

Not Found

Intended User / Care Setting

laboratory professionals

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

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 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.

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.

Summary of Performance Studies

VI Performance Characteristics:

A Analytical Performance:

1. Reproducibility:

a) Reproducibility with Contrived Samples

Study Type: Reproducibility Study
Sample Size: 16 panel members (contrived samples from multiple myeloma cell line spiked into healthy donor whole blood).
Key Results:

Mean fold enrichment: 85.7-fold (low CD138+), 9.3-fold (medium CD138+), 3.8-fold (high CD138+).
Low CD138+ panel members: mean enriched purity 56.2% - 79.6% (from 0.7% - 1.5% unenriched).
Medium CD138+ panel members: mean enriched purity 85.5% - 92.6% (from 7.0% - 12.3% unenriched).
High CD138+ panel members: mean enriched purity 88.5% - 94.0% (from 17.9% - 22.8% unenriched).
All medium and high CD138+ panel members within site and lot had CV 90% of control samples (91.0%, 103.0%, 98.0%). All passed acceptance criteria.
Study 2 (3 clinical specimens, low initial CD138 frequency, 3 replicates per condition): Enriched purities of heparin-spiked specimens were not statistically different from control specimens (one-tailed t-test, p=0.2740). All passed acceptance criteria (>90% of control).
Conclusion: No interference identified from excess anticoagulant (heparin).

4. Stability

Specimen Stability

Study Type: CD138 Expression Stability Study
Sample Size: ~six MM patient BMAs.
Data Source: Multiple myeloma (MM) patient bone marrow aspirates (BMA).
Key Results:

Specimen #1 (initial 10.6%): Enriched purity decreased from 89.9% at baseline to 62.4% at 73 hours. FISH results consistent across time points.
Specimen #2 (initial 0.5%): Purity dropped below LLoQ/LLoD after 44-54 hours. FISH results consistent at baseline and 44-54 hours, but variable/inconclusive for some probes at ~73 hours due to insufficient cells.
Specimen #3 (initial 0.4%): Purity below LLoD at ~73 hours. FISH disposition consistent across time points.
Conclusion: FISH disposition was consistent between baseline and ~73 hours when initial CD138 frequencies were above the flow assay's LLoD.

Kit Stability

Study Type: Shelf-Life and In-Use Stability
Sample Size: Three kit lots for shelf-life, one lot for in-use stability.
Key Results:

Met all acceptance criteria for long-term and in-use storage at 5°C ± 3°C for up to 7 months.
Met acceptance criteria for accelerated storage at 25°C ± 2°C/60% ± 5% RH for up to 7 days.
Kit maintained >95% enriched purity and >70% recovery.
Conclusion: Supports a 6-month shelf life at 5°C ± 3°C and stability for up to 7 days at 25°C ± 2°C/60% ± 5% RH.

Shipping

Study Type: Shipping Stability Study
Sample Size: One batch of EasySep Human Bone Marrow CD138 Positive Selection Kit.
Key Results:

Kits subjected to 48-hour summer (max 30.5°C) and winter (min 3.6°C) temperature profiles performed as expected.
All acceptance criteria (CD138+ cell purity >95% with at least 70% cells recovered) were met at the long-term storage condition of 5°C ± 3°C for up to 7 months.
Conclusion: Transport does not affect product performance. Supports a 6-month shelf-life following transport stress.

5. Detection Limit:

a) Limit of Detection with Contrived Samples

Study Type: Limit of Detection Study
Sample Size: Contrived samples (healthy donor whole blood/bone marrow spiked with CD138+ cell line SK-MM-2) at various initial frequencies (20% down to 0%). Each sample enriched in duplicate.
Key Results:

All contrived specimens spiked with SK-MM-2 cells, targeting as low as 0.08% initial CD138+ frequency, resulted in abnormal t(11:14) FISH disposition.
The average enriched CD138+ purity resulting in abnormal t(11:14) FISH was 33%, from specimens with an average unenriched purity of 0.07%.
The limit of detection (LoD) was determined to be in the range of 0.05 - 0.15% CD138+ measured by flow cytometry. Set at 0.05% CD138+, which is the LoD of the flow cytometer.
Conclusion: The kit consistently enriches from low initial CD138+ frequencies, yielding consistent abnormal FISH patterns down to 0.05% initial frequency.

b) Limit of Detection with Clinical Multiple Myeloma Bone Marrow Specimens

Study Type: Clinical Limit of Detection Study
Sample Size: 5 low CD138 frequency clinical specimens (pre-enrichment 0.2%, 0.5%, 0.7%, 1.5%, 1.7% CD138+).
Key Results:

All 5 specimens successfully enriched to purity ≥ 18.5% CD138+ with fold enrichment > 29.6-fold.
All 5 specimens showed an increase in percentage of abnormal nuclei in FISH analysis for at least one probe.
Three specimens only had abnormal nuclei above FISH cutoff after enrichment.
Conclusion: Clinical MM BM specimens with initial CD138+ frequency near the LoD were successfully enriched and gave interpretable FISH results.

c) Verification of Limit of Detection with Clinical Multiple Myeloma Bone Marrow Specimens

Study Type: LoD Verification Study
Sample Size: One clinical specimen (MM BM aspirate spiked into healthy donor BM aspirate) at 0.05% initial CD138 frequency. Tested using two kit lots with three replicates per lot.
Key Results:

Specimen enriched from 0.05% to a range of 4.1% to 7.4%.
Overall enriched purity CV was 19.7% (passed acceptance criteria of ≤35%).
All enriched samples had FISH disposition consistent with the undiluted clinical specimen (normal for some probes, abnormal for others).
100% of replicates were abnormal for Chromosome 13q deletion and TP53.
Conclusion: Verified the LoD at 0.05% initial CD138 frequency for clinical specimens.

B Clinical Studies:

1. Clinical Study

Study Type: Clinical Study
Sample Size: 33 multiple myeloma patients (bone marrow aspirates, BMA).
Data Source: Bone marrow aspirates from 33 multiple myeloma patients at various stages of disease.
Key Results:

CD138 purity increased from 12.9% ± 15.9% (pre-enriched) to 79.6% ± 19.9% (post-enriched).
Fold enrichment was >1 for all specimens with CD138 frequencies 29.6-fold.
CD138+ Cell Recovery:
- Low initial CD138+ frequency (repeatability): 34.98% - 76.69% (Mean = 53.42%).
- Medium initial CD138+ frequency (repeatability): 36.89% - 65.16% (Mean = 50.62%).
Precision (CV for enriched CD138+ purity):
- Reproducibility CV: High (2.8-6.0%), Med (2.2-7.0%), Low (8.2-18.3%)
- Repeatability (Low freq): 0.81% to 1.56%
- Within-laboratory (Low freq): 0.84% to 1.77%
- Repeatability (Medium freq): 0.17% to 1.41%
- Within-laboratory (Medium freq): 0.65% to 0.96%
Limit of Detection (LoD): 0.05% initial CD138+ frequency (based on flow cytometer LoD).
FISH concordance: Normal/abnormal disposition was concordant between replicates and kit lots. Increased percentage of abnormal nuclei in enriched samples.

Predicate Device(s)

Not Found

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

N/A

Summary

0

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR EasySep Human Bone Marrow CD138 Positive Selection Kit DECISION SUMMARY

I Background Information:

A De Novo Number

DEN220090

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 cell
enrichment 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.

1

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 | Packaging
Configuration | Storage | Content |
|---------------------------------------------------------------------------------|----------------------------|----------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| EasySep Human Bone
Marrow CD138 Positive
Selection Cocktail
(300-0394) | 3 x 1 mL vials | Store at 2 -
8°C. Do
not freeze. | A combination of monoclonal antibodies in
Dulbecco's Phosphate Buffered Saline. Includes an Fc
receptor blocking antibody and Recombinant Human
Albumin stabilizer. |
| EasySep Dextran
RapidSpheres 50105
(300-0400) | 3 x 1 mL vials | Store at 2 -
8°C. Do
not 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 Cell
Lysis Buffer, 10x
Concentrate (100-0749)	1 x 20 mL
Bottle	Store at
15 - 25°C.	A 10x concentrated red blood cell lysis
reagent.
EasySep Buffer
(100-0780)	1 x 1 L	Store at
2 - 8°C. Do not
freeze.	A Dulbecco's Phosphate Buffered Saline
based cell separation buffer.
"The Big Easy" EasySep
Magnet (18001)	1	Ambient temperature	Immunomagnetic column-free magnet.

2

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:

1. 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 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.

3

| CD138+ Initial
Frequency | N | Repeatability
CV (%) | Between Site
CV (%) | Between Lot CV
(%) | Reproducibility
CV (%) | 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 ( 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 CD138

Frequency
(%) | After Enrichment | | CD138 Purity:
Heparin/ PBS
Control | Meets Acceptance
Criteria?
(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 90% of the enriched CD138+ purity from the control samples without excess heparin.

11

| Clinical
Specimen | Target
Initial %
CD138 | Replicate | Enriched Purity
(% CD138+) | | Mean Enriched
Purity (% CD138+) | | Heparin/
Control (%) | Acceptance
Criteria
(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 (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+.

| Target
CD138+
Initial

Frequency	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	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

| MM
Patient
Sample | CD138
Freq* | Pre- Enr
CD138
Purity | Enr
CD138
Purity | Fold
Enr& | % 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 = 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

| MM
Patient
Sample | CD138
Frequency* | Pre-
Enr
CD138
Purity | Enriched
CD138
Purity | Fold
Enrich-
ment& | Probe
(D5S23) | | Probe
(D15Z4) | | Probe
(MYC) | | Probe
(D13S319,
13qter) | | Probe
(IGH/
CCND1
XT) | | Probe
(TP53,
D17Z1) | |
|-------------------------|------- | | Enr | Enr | Pre-
Enr | Enr | Pre-
Enr | Enr | Pre-
Enr | Enr | Pre-
Enr | Enr Enr | Enr |
| 1 | medium | 2 | high | 3 | high | 4 | high | 5 | medium | 6 | medium | 7 | medium | 8 | high | 9 | medium | 10 | high | 11 | medium | 12 | high | 13 | medium | 14 | medium | 15 | medium | 16 | high | 17 | medium | 18 | low | 19 | medium | 20 | high | 21 | low | 22 | low | 23 | high | 24 | medium | 25 | low | 26 | medium | 27 | low | 28 | high | 29 | medium | 30 | low | 31 | medium | 32 | medium | 33 | medium --------------|--------------------------------|-----------------------------|--------------------------|------------------|-----|------------------|-----|----------------|-----|-------------------------------|-----|--------------------------------|------|---------------------------|-----|
| | | | Pre-
| Pre-
| 5.0 | 57.3 | 10.5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 51 | 13 | 53 | 0 | 0 |
| 15.5 | 91.4 | 4.9 | 35.5 | 87 | 21.5 | 63 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 15.6 | 98.2 | 5.3 | 0 | 0 | 0 | 0 | 0 | 17 | 19 | 88 | 0 | 0 | 0 | 0 |
| 82.7 | 77.7 | -0.1 | 38 | 45 | 34.5 | 30 | 69 | 91 | 50.5 | 74 | 0 | 0 | 54 | 70 |
| 4.3 | 57.0 | 12.3 | 25.5 | 74 | 28.5 | 67 | 0 | 0 | 0 | 0 | 0 | 8.5 | 0 | 0 |
| 6.5 | 98.6 | 14.2 | 0 | 0 | 19 | 44 | 25.5 | 46 | 14.5 | 73 | 32.5 | 92 | 26.5 | 77 |
| 10.0 | 94.4 | 8.4 | 20 | 69 | 17.5 | 71 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 36.2 | 93.7 | 1.6 | 45 | 88 | 0 | 0 | 39 | 53 | 0 | 0 | 54 | 94 | 0 | 0 |
| 13.8 | 92.0 | 5.7 | 0 | 0 | 0 | 0 | 29.5 | 89 | 24.5 | 89 | 0 | 0 | 0 | 0 |
| 31.4 | 85.7 | 1.7 | 0 | 0 | 9 | 75 | 0 | 0 | 0 | 90 | 18 | 95.5 | 0 | 0 |
| 7.9 | 85.6 | 9.8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 95 | 9.5 | 91.5 | 0 | 0 |
| 16.2 | 91.0 | 4.6 | 29.5 | 85 | 16 | 51 | 0 | 0 | 41 | 93 | 21.5 | 86.5 | 0 | 0 |
| 3.7 | 88.9 | 23.0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 12.4 | 75.3 | 5.1 | 14.5 | 47 | 0 | 0 | 23.5 | 88 | 24 | 97 | 0 | 0 | 9.5 | 43 |
| 7.7 | 81.7 | 9.6 | 0 | 0 | 17 | 53 | 0 | 6 | 20.5 | 93 | 0 | 8.5 | 0 | 0 |
| 35.0 | 95 | 1.7 | 0 | 0 | 0 | 0 | 0 | 0 | 40 | 88 | 0 | 0 | 46.5 | 81 |
| 4.6 | 82.9 | 17.0 | 0 | 0 | 0 | 0 | 0 | 0 | 18 | 77 | 8 | 82 | 0 | 22 |
| 0.7 | 64.5 | 91.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 85.5 | 0 | 0 |
| 4.9 | 88.3 | 17.0 | 10.5 | 75 | 7 | 27 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 27.7 | 96.8 | 2.5 | 77 | 91 | 68 | 81 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 0.2 | 18.5 | 91.5 | 1.5 | 48 | 0 | 0 | 0 | 0 | 0 | 50 | 0 | 0 | 0 | 0 |
| 1.7 | 52.0 | 29.6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 80 | 0 | 0 | 0 | 0 |
| 17.2 | NC | n/a | 15 | 95 | 16 | 93 | 0 | 0 | 0 | 0 | 29 | 92 | 0 | 0 |
| 5.7 | NC | n/a | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 90 | 0 | 0 | 0 | 0 |
| 2.3 | NC | n/a | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 7 | 64 | 0 | 0 |
| 10.6 | 96.3 | 8.1 | 8.5 | 68 | 9 | 73 | 7.5 | 37 | 0 | 0 | 0 | 0 | 0 | 0 |
| 1.5 | 80.6 | 52.7 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 49 | 0 | 77 | 0 | 0 |
| 20 | 92.1 | 3.6 | 0 | 0 | 4 | 61 | 0 | 0 | 40 | 91 | 0 | 0 | 0 | 0 |
| 9.7 | 90.6 | 8.3 | 21.5 | 75 | 13.5 | 55 | 0 | 8 | 18.5 | 84 | 0 | 0 | 0 | 0 |
| 0.5 | 32.8 | 64.6 | 5 | 53 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 5 | 84.6 | 15.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 83 | 0 | 0 |
| 5.8 | 87.5 | 14.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 80 | 0 | 0 | 0 | 0 |
| 4.3 | 56.2 | 12.1 | 28 | 91 | 28 | 87 | 18 | 72 | 0 | 96 | 0 | 0 | 0 | 0 |

18

Enr = enriched

*low = 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