Predicate Devices

Not Found

Reference Devices

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AI/ML (Artificial Intelligence / Machine Learning)

No
The device description details a standard ELISA assay kit with reagents and controls. There is no mention of AI, ML, or any computational analysis beyond standard laboratory calculations for determining activity levels.

Therapeutic

No
This device is an in vitro diagnostic (IVD) assay used to aid in the diagnosis of a condition by measuring a biomarker, not to treat or directly manage a disease.

Diagnostic

Yes
The "Intended Use / Indications for Use" section explicitly states that the assay is "intended to be used in conjunction with other clinical and laboratory findings as an aid in the diagnosis of thrombotic thrombocytopenic purpura (TTP)".

SaMD (Software as a Medical Device)

No

The device description clearly outlines multiple physical components including a microplate, reagents, calibrators, controls, conjugate, substrate, and stop solution, indicating it is a hardware-based assay kit.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The intended use explicitly states it's for the "qualitative determination of ADAMTS13 activity in platelet poor human citrated plasma." This involves testing a biological sample (plasma) outside of the body.
Device Description: The description details an "enzyme-linked immunosorbent assay (ELISA)" which is a common laboratory technique used for in vitro testing. It also lists reagents and components used to perform this test on a plasma sample.
Clinical Performance Study: The description of the clinical performance study involves testing "residual samples selected from a local repository of frozen human citrated plasma." This further confirms the testing is performed on biological samples outside the body.

All of these points align with the definition of an In Vitro Diagnostic device, which is used to examine specimens derived from the human body to provide information for diagnostic purposes.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Technozvm ADAMTS13 Activity assay is an enzyme-linked immunosorbent assay (ELISA) intended for the qualitative determination of ADAMTS13 activity in platelet poor human citrated plasma. The assay is intended to be used in conjunction with other clinical and laboratory findings as an aid in the diagnosis of thrombotic thrombocytopenic purpura (TTP) in adult and pediatric patients being evaluated for thrombotic microangiopathy (TMA).

Product codes (comma separated list FDA assigned to the subject device)

SAC

Device Description

The Technozym ADAMTS13 Activity assay is an enzyme linked immunosorbent assay (ELISA) used for detection of ADAMTS13 activity in citrated human plasma.

The assay contains:

ADAMTS13 Activity anti-GST coated test plate microplate coated with anti-GST antibody .
ADAMTS13 Activity GST-VWF73 reagent that contains GST tagged peptide of 73 amino . acids from the A2 domain of VWF with specific cleavage site for ADAMTS13 and serves as the in vitro substrate for ADAMTS13
ADAMTS13 Activity Calibrators-consists of six vials containing lyophilized plasma, each . with a different level of ADAMTS13 activity
ADAMTS13 Activity Controls consists of two vials of lyophilized plasma, each with high . or low levels of ADAMTS13 activity
. ADAMTS13 Activity Conjugate - reagent that contains horseradish peroxidase (HRP) conjugated monoclonal antibody directed against the neoepitope exposed due to cleavage of GST-VWF73 by ADAMTS13 present in plasma
ADAMTS13 TMB substrate reagent contains tetramethylbenzidine (TMB) substrate for . HRP
ADAMTS13 Activity Stop Solution reagent contains 2.5% sulfuric acid for stopping the . conversion of TMB substrate

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

adult and pediatric patients

Intended User / Care Setting

Not Found

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

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision/Reproducibility:
Studies were conducted using quality controls and nine human plasma sample pools.
Within-laboratory precision: Each sample was tested for five days with two runs per day and two replicates per run at a single site using three reagent lots for a total of 30 replicate measurements per sample. Samples tested included levels below, around and above the assay cut-off of 0.1 IU/mL.
Operator-to-operator precision: The study was conducted over five days using one reagent lot with two runs per day and two replicates per run by three operators for a total of 30 mean results per sample level. The study design included six samples prepared by mixing plasma from normal human donors with native deficient plasma (TTP patient plasma) in different ratios. In addition, three sample levels were prepared by mixing plasma from normal human donors with heat inactivated plasma.
Site-to-site reproducibility: The study was performed at three study sites. At each site, the samples were assayed on each of five days, with two runs per day and two replicates per run, using one lot of reagents, resulting in a total of 30 mean results per sample level. The study design included six sample levels prepared by mixing plasma from normal human donors with native deficient plasma (TTP patient plasma) in different ratios. In addition, three sample levels were prepared by mixing plasma from normal human donors with heat inactivated plasma.

Analytical Specificity/Interference:
Interference studies were conducted by paired-difference testing using one lot of reagents for both common endogenous and extrinsic interferents. Each sample was tested in five replicates. Samples with and without the interferent were measured, and the measurand concentration difference was determined. None of the listed substances led to clinically significant interference.

Clinical Performance Study:
The clinical performance study was conducted at two external sites (one in U.S., one outside U.S.). Testing was performed double blinded. Study samples were residual samples selected from a local repository of frozen human citrated plasma from patients diagnosed with thrombotic microangiopathies (TMA), from donors > 6 months of age.
Sample size: 137 samples.
Key results:
Sensitivity = 84.8% (28/33); 95% CI: (69.1% to 93.3%)
Specificity = 97.1% (101/104); 95% CI: (91.9% to 99%)
Positive Predictive Value (PPV) = 90.2% (28/31); 95% CI: (75.2% to 96.6%)
Negative Predictive Value (NPV) = 95.3% (101/106); 95% CI: (90.0% to 97.8%)

Other Supportive Performance Characteristics Data:
Prozone Effect (Hook Effect): No significant hook effect was observed up to activity levels of 8 IU/mL.
Cross-contamination Studies: No cross-contamination was observed.
Reagent Stability Studies: Real-time shelf-life stability supports 24 months at 2-8°C.
Sample Stability Studies: Frozen sample stability of 12 months. Fresh Sample stability up to 8 hours at room temperature (18-25°C) and up to 24 hours under refrigerated conditions (2-8°C).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Sensitivity = 84.8% (28/33); 95% CI: (69.1% to 93.3%)
Specificity = 97.1% (101/104); 95% CI: (91.9% to 99%)
Positive Predictive Value (PPV) = 90.2% (28/31); 95% CI: (75.2% to 96.6%)
Negative Predictive Value (NPV) = 95.3% (101/106); 95% CI: (90.0% to 97.8%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not Found

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

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Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

N/A

Summary

0

Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: a symbol on the left and the text "FDA U.S. FOOD & DRUG ADMINISTRATION" on the right. The symbol is a stylized representation of a caduceus, a traditional symbol of medicine. The text is in a combination of blue and black, with "FDA" in blue and the rest of the text in black.

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Technozym ADAMTS13 Activity DECISION SUMMARY

Background Information: I

A De Novo Number

DEN230024

B Applicant

Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH

C Proprietary and Established Names

Technozym ADAMTS13 Activity

D Regulatory Information

| Product
Code(s) | Classification | Regulation
Section | Panel |
|--------------------|-----------------------------------|-----------------------|------------|
| SAC | Class II with
special controls | 21 CFR 864.7297 | Hematology |

Submission/Device Overview: II

A Purpose for Submission:

De Novo request for evaluation of automatic class III designation for Technozym ADAMTS13 Activity

B Measurand:

ADAMTS13 Activity

C Type of Test:

Manual enzyme linked immunosorbent assay (ELISA)

Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov

1

III Indications for Use:

A Intended Use(s):

See Indications for Use below

B Indication(s) for Use:

The Technozvm ADAMTS13 Activity assay is an enzyme-linked immunosorbent assay (ELISA) intended for the qualitative determination of ADAMTS13 activity in platelet poor human citrated plasma. The assay is intended to be used in conjunction with other clinical and laboratory findings as an aid in the diagnosis of thrombotic thrombocytopenic purpura (TTP) in adult and pediatric patients being evaluated for thrombotic microangiopathy (TMA).

C Special Conditions for Use Statement(s):

For Prescription Use Only For In Vitro Diagnostic Use Only

D Special Instrument Requirements:

Microplate reader

IV Device/System Characteristics:

A Device Description:

The Technozym ADAMTS13 Activity assay is an enzyme linked immunosorbent assay (ELISA) used for detection of ADAMTS13 activity in citrated human plasma.

The assay contains:

ADAMTS13 Activity anti-GST coated test plate microplate coated with anti-GST antibody .
ADAMTS13 Activity GST-VWF73 reagent that contains GST tagged peptide of 73 amino . acids from the A2 domain of VWF with specific cleavage site for ADAMTS13 and serves as the in vitro substrate for ADAMTS13
ADAMTS13 Activity Calibrators-consists of six vials containing lyophilized plasma, each . with a different level of ADAMTS13 activity
ADAMTS13 Activity Controls consists of two vials of lyophilized plasma, each with high . or low levels of ADAMTS13 activity
. ADAMTS13 Activity Conjugate - reagent that contains horseradish peroxidase (HRP) conjugated monoclonal antibody directed against the neoepitope exposed due to cleavage of GST-VWF73 by ADAMTS13 present in plasma
ADAMTS13 TMB substrate reagent contains tetramethylbenzidine (TMB) substrate for . HRP
ADAMTS13 Activity Stop Solution reagent contains 2.5% sulfuric acid for stopping the . conversion of TMB substrate

2

B Test Principle

ADAMTS13, a disintegrin and metalloprotease with thrombospondin type 1 motif 13, is an enzyme (VWF-cleaving protease) that specifically cleaves yon Willebrand factor (VWF) under high shear stress conditions. The Technozym ADAMTS13 Activity assay is an enzyme linked immunosorbent assay for the detection of ADAMTS13 activity in human citrated plasma. GST-VWF73, a substrate that can be specifically cleaved by ADAMTS13 in vitro, is immobilized on to wells of a microplate that is pre-coated with an antibody specific to glutathione S-transferase (GST). After washing away unbound GST-VWF73, samples (i.e., clinical specimens, controls, and calibrators) are pipetted into wells and incubated with immobilized GST-VWF73. ADAMTS13 present in the samples cleaves the VWF73 peptide of immobilized GST-VWF73 at specific sites, exposing the neoepitope on VWF73. After washing away the excess sample, a second mouse monoclonal antibody specific to the neoepitope on GST-VWF73 that has been conjugated to the enzyme horseradish peroxidase (HRP) is added to the well. After washing away unbound HRP-conjugated antibody, the chromogenic substrate is added to the well. The HRP enzyme catalyzes a specific reaction with the chromogenic substrate, which produces a colored product that is detected as absorbance measurement (optical density, OD) at 450 nm with a microplate reader. The amount of absorbance (OD) generated is proportional to ADAMTS13 activity in the well. The results for the wells containing calibrators are used to create a reference curve to quantify the ADAMTS13 activity in the sample.

In line with the recommendation of the International Society of Thrombosis and Haemostasis (ISTH) in the Journal of Thrombosis and Haemostasis (2020), the assay results should be interpreted at the ADAMTS13 Activity assay cut-off of 0.1 IU/mL for thrombotic thrombocvtopenic purpura (TTP). Technozym ADAMTS13 Activity assay results > 0.1 IU/mL will be TTP negative and results ≤ 0.1 IU/mL will be TTP positive. The ADAMTS13 Activity assay results should be interpreted in conjunction with other clinical and laboratory findings.

V Standards/Guidance Documents Referenced:

CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures: Approved Guideline - Third Edition

CLSI EP06-A2: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline - Second Edition

CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition

CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures: Approved Guideline - Second Edition

CLSI EP25-A: Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline

3

Performance Characteristics: VI

Analytical Performance:

1. Precision/Reproducibility:

Precision studies were conducted according to recommendations in CLSI EP05-A3 using quality controls and nine human plasma sample pools, which were prepared by mixing human plasma from normal donors with clinical samples from patients diagnosed with thrombotic thrombocytopenic purpura (TTP) and deficient in ADAMTS13 activity or heat inactivated plasma.

Within-laboratory precision

To evaluate the within-laboratory precision, each sample was tested for five days with two runs per day and two replicates per run at a single site, using three reagent lots for a total of 30 replicate measurements per sample. The samples tested included levels below, around and above the assay cut-off of 0.1 IU/mL. The quantitative results are summarized in the tables below.

| Sample | N | Mean
(IU/mL) | Repeatability | | Between-run | | Between-day | | Between-lot | | Within-
laboratory | |
|--------|----|-----------------|---------------|------|-------------|------|-------------|------|-------------|------|-----------------------|------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| S1 | 30 | 0.65 | 0.03 | 4.46 | 0.03 | 5.10 | 0.02 | 3.60 | 0.00 | 0.00 | 0.05 | 7.66 |
| S2 | 30 | 0.45 | 0.01 | 2.26 | 0.04 | 8.50 | 0.00 | 0.00 | 0.01 | 3.00 | 0.04 | 9.26 |
| S3 | 29 | 0.24 | 0.01 | 3.25 | 0.01 | 2.50 | 0.00 | 1.00 | 0.00 | 1.70 | 0.01 | 4.52 |
| S4 | 29 | 0.19 | 0.00 | 1.84 | 0.01 | 4.20 | 0.00 | 0.00 | 0.00 | 2.10 | 0.01 | 4.97 |
| S5 | 30 | 0.14 | 0.00 | 2.19 | 0.01 | 4.90 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | 5.17 |
| S6 | 30 | 0.08 | 0.00 | 2.05 | 0.01 | 8.10 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | 7.87 |
| S7 | 30 | 0.65 | 0.03 | 4.88 | 0.03 | 3.80 | 0.02 | 2.90 | 0.01 | 0.90 | 0.05 | 6.90 |
| S8 | 30 | 0.23 | 0.01 | 3.04 | 0.01 | 5.40 | 0.00 | 0.00 | 0.00 | 0.80 | 0.01 | 6.24 |
| S9 | 30 | 0.12 | 0.00 | 2.62 | 0.01 | 4.10 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | 4.91 |

| Sample | Mean
(IU/mL) | Total
results | Qualitative agreement | |
|----------------|-----------------|------------------|------------------------------|----------------|
| | | | Number of correct
results | % Correct call |
| S1
negative | 0.65 | 30 | 30/30 | 100 |
| S2
negative | 0.45 | 30 | 30/30 | 100 |
| S3
negative | 0.24 | 29 | 29/29 | 100 |
| S4
negative | 0.19 | 29 | 29/29 | 100 |
| S5
negative | 0.14 | 30 | 30/30 | 100 |

4

| Sample | Mean
(IU/mL) | Total
results | Qualitative agreement | |
|----------------|-----------------|------------------|------------------------------|----------------|
| | | | Number of correct
results | % Correct call |
| S6
positive | 0.08 | 30 | 30/30 | 100 |
| S7
negative | 0.65 | 30 | 30/30 | 100 |
| S8
negative | 0.23 | 30 | 30/30 | 100 |
| S9
negative | 0.12 | 30 | 30/30 | 100 |

Operator-to-operator

The study was conducted over five days using one reagent lot with two runs per day and two replicates per run by three operators for a total of 30 mean results per sample level. The study design included six samples prepared by mixing plasma from normal human donors with native deficient plasma (TTP patient plasma) in different ratios. In addition, three sample levels were prepared by mixing plasma from normal human donors with heat inactivated plasma. The samples tested included levels below, around and above the assay cut-off of 0.1 IU/mL.

Sample	N	Mean (IU/mL)	Repeatability		Between-run		Between-day		Between-operator		Within-laboratory
			SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
S1	30	0.67	0.03	4.17	0.03	5.10	0.03	4.80	0.01	1.90	0.06	8.27
S2	30	0.45	0.01	2.26	0.04	9.70	0.00	0.00	0.01	1.30	0.05	10.08
S3	29	0.24	0.01	2.90	0.01	4.30	0.00	0.00	0.01	2.30	0.01	5.59
S4	29	0.19	0.00	2.19	0.01	5.40	0.00	0.00	0.00	2.20	0.01	6.10
S5	30	0.13	0.00	1.77	0.01	3.60	0.01	3.40	0.00	2.50	0.01	5.99
S6	30	0.07	0.00	2.69	0.00	6.00	0.00	0.00	0.00	4.10	0.01	8.12
S7	30	0.65	0.03	4.81	0.03	4.80	0.00	0.00	0.01	1.80	0.05	7.11
S8	30	0.23	0.01	2.26	0.01	4.90	0.00	1.70	0.00	1.60	0.01	5.84
S9	30	0.12	0.00	2.05	0.01	5.20	0.00	1.20	0.00	2.40	0.01	6.18

| Sample | Mean
(IU/mL) | Total
results | Qualitative agreement | |
|----------------|-----------------|------------------|------------------------------|----------------|
| | | | Number of correct
results | % Correct call |
| S1
negative | 0.67 | 30 | 30/30 | 100 |
| S2
negative | 0.45 | 30 | 30/30 | 100 |
| S3
negative | 0.24 | 29 | 29/29 | 100 |
| S4
negative | 0.19 | 29 | 29/29 | 100 |

5

| Sample | Mean
(IU/mL) | Total
results | Qualitative agreement | |
|----------------|-----------------|------------------|------------------------------|----------------|
| | | | Number of correct
results | % Correct call |
| S5
negative | 0.13 | 30 | 30/30 | 100 |
| S6
positive | 0.07 | 30 | 30/30 | 100 |
| S7
negative | 0.65 | 30 | 30/30 | 100 |
| S8
negative | 0.23 | 30 | 30/30 | 100 |
| S9
negative | 0.12 | 30 | 30/30 | 100 |

Site-to-site reproducibility

The study was performed at three study sites. At each site, the samples were assayed on each of five days, with two runs per day and two replicates per run, using one lot of reagents, resulting in a total of 30 mean results per sample level. The study design included six sample levels prepared by mixing plasma from normal human donors with native deficient plasma (TTP patient plasma) in different ratios. In addition, three sample levels were prepared by mixing plasma from normal human donors with heat inactivated plasma. To prepare heat inactivated plasma with no residual ADAMTS13 activity, citrated plasma samples from normal donors were heat-inactivated for 1 hour at 56℃. The samples tested included levels below, around and above the cut-off of 0.1 IU/mL.

Sample	N	Mean (IU/mL)	Repeatability		Between-run		Between-day		Between-site		Reproducibility
			SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
S1	30	0.67	0.03	4.81	0.04	6.20	0.05	6.60	0.00	0.00	0.07	10.24
S2	30	0.46	0.02	4.67	0.04	8.40	0.00	0.00	0.01	2.20	0.05	9.86
S3	29	0.25	0.01	3.75	0.02	6.60	0.00	0.00	0.01	4.80	0.02	8.99
S4	29	0.19	0.01	4.46	0.02	7.40	0.00	0.00	0.00	0.00	0.02	8.64
S5	30	0.14	0.01	4.17	0.01	9.00	0.00	0.00	0.00	0.00	0.01	9.88
S6	30	0.07	0.00	4.53	0.01	6.30	0.00	0.00	0.01	7.00	0.01	10.45
S7	30	0.66	0.03	4.46	0.04	5.70	0.01	2.00	0.03	3.90	0.06	8.49
S8	30	0.23	0.01	4.31	0.01	5.30	0.00	0.00	0.00	0.00	0.02	6.85
S9	30	0.12	0.00	2.97	0.01	6.40	0.00	0.00	0.00	3.60	0.01	8.01

Sample	Mean (IU/mL)	Total results	Qualitative agreement
			Number of correct results	% Correct call
S1 negative	0.67	30	30/30	100
S2 negative	0.46	30	30/30	100

6

	Mean	Total	Qualitative agreement
Sample	(IU/mL)	results	Number of correct
results	% Correct call
S3
negative	0.25	29	29/29	100
S4
negative	0.19	29	29/29	100
S5
negative	0.14	30	30/30	100
S6
positive	0.07	30	30/30	100
S7
negative	0.66	30	30/30	100
S8
negative	0.23	30	30/30	100
S9
negative	0.12	30	30/30	100

2. Analytical Specificity/Interference:

Interference studies were conducted based on the CLSI EP07 3rd Edition guideline. Three base pools mimicking high (1.0 IU/mL), medium (0.5 IU/mL) and low (0.1 IU/mL) levels of ADAMTS13 activity were prepared by mixing human citrated plasma (non-icteric, nonturbid and non-hemolyzed) with plasma rendered ADAMTS13 deficient by heat inactivation. Interference testing was conducted by paired-difference testing using one lot of reagents for both common endogenous and extrinsic interferents. Each sample was tested in five replicates. Samples with and without the interferent were measured, and the measurand concentration difference was determined.

None of the substances in the following table were found to lead to clinically significant interference.

| Potential interfering
substance | No interference up to the
following evaluated clinically
significant concentration: |
|------------------------------------|-------------------------------------------------------------------------------------------|
| Exogenous | |
| Acetaminophen | 15.6 mg/dL |
| Acetylcysteine | 15.0 mg/dL |
| Ampicillin Na | 7.5 mg/dL |
| ASA | 3.0 mg/dL |
| Biotin | 0.351 mg/dL |
| Caplacizumab | 0.15 mg/dL |
| Cefoxitin Na | 660.0 mg/dL |
| Cyclosporine | 0.18 mg/dL |
| Doxycycline | 1.8 mg/dL |
| Heparin | 330 units/dL |
| Ibuprofen | 21.9 mg/dL |
| Levodopa | 0.75 mg/dL |
| Methyldopa | 2.25 mg/dL |

7

| Potential interfering
substance | No interference up to the
following evaluated clinically
significant concentration: |
|------------------------------------|-------------------------------------------------------------------------------------------|
| Exogenous | |
| Metronidazole | 12.3 mg/dL |
| Phenylbutazone | 32.0 mg/dL |
| Prednisolone | 0.12 mg/dL |
| Rifampicin | 4.8 mg/dL |
| Rituximab | 50.0 mg/dL |
| Theophylline | 6.0 mg/dL |
| Endogenous | |
| Intralipid | 500 mg/dL |
| Hemoglobin | 220 mg/dL |
| Unconjugated Bilirubin | 66.0 mg/dL |
| Conjugated Bilirubin | 66.0 mg/dL |
| GST | 0.02 mg/dL |
| VWF | 2.0 IU/mL |
| Human anti mouse antibody | titer >12 |
| Rheumatoid factor | 156 IU/mL |

3. Assay Reportable Range:

Not applicable

1. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

Traceability

Target values for calibrators and controls are traceable to the first International Standard for ADAMTS13 Activity and Antigen in Plasma (NIBSC WHO 1st international Standard ADAMTS13 Plasma 12/252).

Stability of calibrators and controls

Stability of calibrators and controls were evaluated in accordance with CLSI EP25A. Three lots of calibrators and controls were used in the study and stored in their final packaging at 2-8°C. At time points 0, 12, 24 and 30 months, sets of calibrators and controls were placed into stable storage (-70°C). At the end of the study (t=30 months), all calibrators and controls were tested in triplicate in one single run on one instrument using one lot of reagents. The data supported a real-time stability of 24 months.

1. Assay Cut-Off:
  Not applicable

8

B Comparison Studies:

1. Clinical Performance Study:

The clinical performance study was conducted at two external sites, one located in U.S. and the other located outside of the U.S. Testing was performed double blinded. The clinician making the diagnosis decisions and selecting the samples was blinded to the Technozym ADAMTS 13 activity results and the laboratory technician conducting the Technozym assay was blinded to the diagnosis. Samples were tested in duplicate using the Technozym ADAMTS13 Activity assay. One kit lot was used per study site. At each study site, tests were performed by one laboratory professional. The study samples used in testing were residual samples selected from a local repository of frozen human citrated plasma from patients diagnosed with thrombotic microangiopathies (TMA) (i.e., clinical suspicion of thrombotic thrombocytopenic purpura (TTP)) by board-certified clinician according to the local testing algorithm for TMAs. All patient samples were from donors > 6 months of age and patient population is representative of intended use population.

Combined agreement analysis for both sites with a total of 137 samples included in the clinical performance study.

		Clinical diagnosis of TTP
		Positive	Negative	Total
Technozym
ADAMTS13
Activity	Positive	28	3	31
	Negative	5	101	106
Total		33	104	137
	Sensitivity = 84.8% (28/33); 95% CI: (69.1% to 93.3%)
Specificity = 97.1% (101/104); 95% CI: (91.9% to 99%)		Positive Predictive Value (PPV) = 90.2% (28/31); 95% CI: (75.2% to 96.6%)
Negative Predictive Value (NPV) = 95.3% (101/106); 95% CI: (90.0% to 97.8%)

C Clinical Studies:

1. Clinical Sensitivity:
  Refer to Clinical Performance Study

2. Clinical Specificity:

Refer to Clinical Performance Study

1. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
  Not applicable

9

D Clinical Cut-Off:

The clinical cut-off for TTP diagnosis is 10% or 0.1 IU/mL ADAMTS13 activity.

E Other Supportive Performance Characteristics Data:

1. Prozone Effect (Hook Effect)

Information was provided to support that no significant hook effect was observed up to activity levels of 8 IU/mL.

2. Cross-contamination Studies

A study was performed to evaluate if cross-contamination and/or carryover occurs between samples in the plate wells during the assay procedure. Low samples with a target concentration of 0.1 IU/mL and high samples with a target concentration of 1.0 IU/mL were used to perform the studies. In the first stage, the signal of only low samples was evaluated throughout the microplate. In the second stage, two test plates were run with an alternating pattern over all available patient sample locations. The pattern consisted of two wells containing only the low samples followed by two wells containing the high sample. The study was performed by three operators performing testing with three microplate readers and plate washer combinations with one lot of reagents. No cross-contamination was observed.

3. Reagent Stability Studies:

Real-time Shelf-life Stability Studies

The real-time stability study was conducted in accordance with CLSI guideline EP25-A. The study was conducted with three lots of Technozym ADAMTS13 Activity assay kits. Eight samples were prepared by mixing citrated human plasma in human ADAMTS13 activity deficient plasma (HIP) in different ratios. These samples were aliquoted and frozen at -20℃ and a fresh aliquot was used for every test time point. Reagent kits were stored in their final packaging at 2-8°C. Time points used in the real time stability study included: 0, 6. 12, 24 and 30 months. Reagent kits were retrieved and tested with different ADAMTS13 activity sample levels at the end of each designated time point in the study. The testing was done in duplicates for each ADAMTS13 activity level. Based on the real-time stability results, the data supports a shelf-life of the Technozym ADAMTS13 Activity assay kit for up to 24 months at 2-8°C.

4. Sample Stability Studies

Frozen sample stability

Eight samples were prepared by mixing citrated human plasma with heat treated citrated human plasma in different ratios, and aliquots was stored frozen at