The CRYOcheck FVIII Inhibitor Kit is for clinical laboratory use in conjunction with a Factor VIII activity assay to enable the performance of a modified Nijmegen-Bethesda assay using 3.2% citrated human plasma. It enables the determination of a functional FVIII inhibitor titer to aid in the clinical management of congenital hemophilia A in individuals aged 2 years or older. For in vitro diagnostic use.

Device Description

The FVIII Inhibitor Kit is used in the CDC modification of the Nijmegen-Bethesda assay and contains the following components:
Imidazole Buffered Pooled Normal Plasma (IB-PNP): Pooled normal plasma from a minimum of twenty donors with a factor VIII activity value of 95-113% and buffered with imidazole to a pH of 7.3 – 7.5.
Imidazole Buffered Bovine Serum Albumin (IB-BSA): A 4% BSA solution buffered with imidazole to a pH of 7.3-7.5.
Negative Factor VIII Inhibitor Control: Pooled normal plasma from a minimum of five donors buffered with HERES to a pH of 6.2-8.2
Positive Factor VIII Inhibitor Control: HEPES buffered (pH 6.2-8.2) immunodepleted FVIII deficient plasma to which anti-human FVIII antibodies have been added.

AI/ML Overview

The document provided describes the performance characteristics and studies for the CRYOcheck FVIII Inhibitor Kit, rather than an AI/ML-driven medical device. Therefore, a direct mapping to all the requested criteria for AI/ML device acceptance (such as MRMC studies, number of experts for ground truth, adjudication methods, and training set details) is not possible.

However, I can extract and present the information relevant to the device's performance and the studies conducted to demonstrate its effectiveness, aligning with the spirit of the acceptance criteria for a diagnostic kit.

Device: CRYOcheck™ FVIII Inhibitor Kit

Intended Use: For clinical laboratory use in conjunction with a Factor VIII activity assay to enable the performance of a modified Nijmegen-Bethesda assay using 3.2% citrated human plasma. It enables the determination of a functional FVIII inhibitor titer to aid in the clinical management of congenital hemophilia A in individuals aged 2 years or older. For in vitro diagnostic use.

Acceptance Criteria and Reported Device Performance

Since this is an in-vitro diagnostic kit and not an AI/ML device, the "acceptance criteria" are related to standard analytical performance characteristics. The table below summarizes the key performance metrics and the reported results.

Acceptance Criterion (Performance Metric)	Reported Device Performance
Precision (Multi-Reagent Lot)	- Kit Negative Control: SD 0.1 BU/mL (304.1% CV) - Kit Positive Control: 9.4% CV - Negative Plasma Sample: SD 0.1 BU/mL (24.4% CV) - Low Plasma Sample: 8.2% CV - Mid Plasma Sample: 9.9% CV - High Plasma Sample: 6.7% CV (Aggregated Data for all 3 lots) - Negative Plasma Sample: SD 0.1 BU/mL (29.0% CV) - Low Plasma Sample: 8.2% CV - Mid Plasma Sample: 8.3% CV - High Plasma Sample: 8.4% CV
Reproducibility (Multi-Reagent Lot Site to Site)	- Kit Negative Control: SD 0.1 BU/mL (422.7% CV) - Kit Positive Control: 16.0% CV - Negative Plasma Sample: SD 0.1 BU/mL (28.4% CV) - Low Plasma Sample: 10.6% CV - Mid Plasma Sample: 8.3% CV - High Plasma Sample: 13.2% CV (Pooled 3-Site Data) The results demonstrated a pooled reproducibility of ≤16% CV for the positive samples and 0.1 BU/mL SD for the negative plasma sample.
Linearity/Assay Reportable Range	Linearity Range: 0.2 to 1402.9 BU/mL.
Shelf-Life Stability	Supported a 12-month shelf-life stability claim when stored at <-70°C.
In-Use Stability	Supported a 4-hour in-use stability of the product when maintained at room temperature (18–25 °C) or at 2-8 °C post thaw.
Detection Limit	- Limit of Blank (LoB): 0.1 BU/mL - Limit of Detection (LoD): 0.2 BU/mL - Limit of Quantitation (LoQ): 0.2 BU/mL
Interference	No interference found with: hemoglobin (≤ 500mg/dL), bilirubin (≤ 500mg/dL), Intralipid (≤ 29mg/dL), vWF (≤ 20 µg/mL). Lupus anticoagulant autoantibodies may interfere with the quantification of low titer FVIII inhibitors. Rheumatoid Factor at ≥ 82 IU/mL showed interference.
Method Comparison	- Positive Agreement: 100% (95% Cl, 97-100%) - Negative Agreement: 99% (95% CI, 93-100%) Regression statistics showed equivalent performance to comparator method. Overall Pearson Correlation Coefficient: 0.970 ($r^2$ = 0.940).
Sample Integrity	- Robustness demonstrated for FVIII measurements in fresh and frozen plasma samples, including after multiple freeze/thaw cycles (-80°C to 2-8°C, -80°C to -20°C for up to 15 days, and up to three freeze/thaw cycles at -20°C and -80°C). - Sample stability of 12 months when stored frozen at <-70°C.

Study Details (Relevant to an In-Vitro Diagnostic Kit)

The document describes a series of analytical performance studies for the CRYOcheck FVIII Inhibitor Kit.

Sample Size and Data Provenance:
- Precision and Reproducibility Studies: Four plasma samples from congenital hemophilia A patients (negative, low, mid, high levels of FVIII inhibitor) and the kit's Positive and Negative Controls were used.
  - Precision: 80 replicates per sample per lot (3 lots).
  - Reproducibility: 30 replicates per sample per site (3 sites).
  - Data provenance: Implied to be from internal and external laboratories, likely within North America given the submitter and FDA context. The studies are retrospective analyses of samples.
- Linearity Study: Twelve sample dilutions (0 to 100 BU/mL) created from FVIII inhibitor positive plasma and FVIII inhibitor-negative plasma. An expanded study used eighteen sample dilutions (0 to 1402.9 BU/mL) from a high titer contrived sample.
- Stability Studies: Five to six replicates of kit Positive/Negative Controls and four or three plasma samples (negative, low, mid, high FVIII inhibitor levels).
- Detection Limit Studies: Four blank plasma samples from normal healthy donors (for LoB), and four low titer plasma samples from congenital hemophilia A donors (for LoD). Measured in triplicate using three lots over five days.
- Interference Studies: Patient plasma samples spiked with possible interferents. 10 replicates tested alongside 10 replicates of blank matrix control.
- Method Comparison Studies (Test Set): 210 human plasma samples from individuals with congenital hemophilia A. Aliquots distributed across three sites.
  - Data Provenance: Not explicitly stated, but samples are from congenital hemophilia A patients, suggesting clinical samples from a relevant population. The study is prospective in terms of testing collected samples, but the samples themselves are retrospective in terms of their collection relative to the study.
- Sample Integrity Studies: Three test plasma samples created by spiking anti-FVIII monoclonal antibody into immunodepleted FVIII deficient plasma (negative, low, high FVIII inhibitor levels). A retrospective analysis on four plasma samples from congenital hemophilia A patients.
Number of Experts and Qualifications: Not applicable for this type of IVD kit study. The performance is assessed against a comparator method or internal validation standards, not through expert interpretation of images or other subjective data.
Adjudication Method (for Test Set): Not applicable. This is a quantitative diagnostic test where results are numerical values. The comparison is done against a "central reference laboratory using a chromogenic CDC-Modified Nijmegen-Bethesda Assay which has been implemented as a validated Laboratory-Developed Test."
Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study: Not applicable to an in-vitro diagnostic kit where human interpretation is not the primary output being evaluated. The kit measures a biomarker, and its performance is evaluated based on analytical characteristics.
Standalone Performance: The performance data presented (precision, linearity, detection limits, stability, interference, and method comparison) represent the "algorithm only" or "device only" performance without human-in-the-loop performance, as it's an automated laboratory test.
Type of Ground Truth:
- For analytical performance (precision, linearity, stability, detection limits): "Ground truth" is established by the known concentrations/characteristics of controls, spiked samples, or by reference to established metrological protocols (e.g., CLSI guidelines).
- For method comparison studies: The ground truth was established by a "validated Laboratory-Developed Test" (LDT) based on a chromogenic CDC-Modified Nijmegen-Bethesda Assay performed at a central reference laboratory. This serves as the reference method.
- For sample integrity studies: The "ground truth" for the spiked samples was their known composition or assigned values based on fresh measurements. For the retrospective analysis, the true values would be the FVIII inhibitor titers measured by the established method.
Sample Size for Training Set: Not applicable. This is not an AI/ML device that undergoes a "training" phase. The kit's components and assay methodology are developed through R&D, not machine learning from a specific dataset.
How Ground Truth for Training Set Was Established: Not applicable (see point 7).

Summary

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March 12, 2019

Precision BioLogic Inc. Karen Black VP of Compliance & Product Development 140 Eileen Stubbs Avenue Dartmouth, Nova Scotia B3B 0A9 Canada

Re: K183440

Trade/Device Name: CRYOcheck FVIII Inhibitor Kit Regulation Number: 21 CFR 864.7290 Regulation Name: Factor deficiency test Regulatory Class: Class II Product Code: GGP Dated: December 10, 2018 Received: December 12, 2018

Dear Karen Black:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

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statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.html; good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Leonthena R. Carrington -S

Lea Carrington Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K183440

Device Name CRYOcheck FVIII Inhibitor Kit

Indications for Use (Describe)

Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) Summary cryocheck FVIII Inhibitor Kit

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is: ___K183440

Submitter'sInformation	Precision BioLogic Inc.140 Eileen Stubbs Ave.Dartmouth, Nova Scotia B3B 0A9Canada
Contact Person	Karen M. Black, VP of Compliance & Product DevelopmentPhone: 902-468-6422, ext. 226, or 902-706-3125E-mail: kblack@precisionbiologic.com
Preparation Date	December 10, 2018
Device Trade Name	CRYOcheck™ Factor VIII Inhibitor Kit
RegulatoryInformation	Regulation Number andDescription	21 CFR 864.7290Factor Deficiency Test21 CFR 864.5425Multipurpose system for in vitrocoagulation studies
	Classification	Class II
	Product Code	GGP; Test, Qualitative and QuantitativeFactor Deficiency; 21 CFR 864.7290;GGN; Plasma, Coagulation Control; 21 CFR 864.5425
	Classification Panel	Hematology
	Predicate Device	Immucor FVIII Antibody Screen Assay (K082205)
Indication for Use/Intended Use	The CRYOcheck FVIII Inhibitor Kit is for clinical laboratory use inconjunction with a Factor VIII activity assay to enable performance of amodified Nijmegen-Bethesda assay using 3.2% citrated human plasma.It enables the determination of a functional FVIII inhibitor titer to aid inthe clinical management of congenital hemophilia A in individuals aged2 years or older. For in vitro diagnostic use.
Device Description	The FVIII Inhibitor Kit is used in the CDC modification of the Nijmegen-Bethesda assay and contains the following components:Imidazole Buffered Pooled Normal Plasma (IB-PNP) : Pooled normalplasma from a minimum of twenty donors with a factor VIII activityvalue of 95-113% and buffered with imidazole to a pH of 7.3 – 7.5.Imidazole Buffered Bovine Serum Albumin (IB-BSA) : A 4% BSA solutionbuffered with imidazole to a pH of 7.3-7.5.Negative Factor VIII Inhibitor Control : Pooled normal plasma from aminimum of five donors buffered with HERES to a pH of 6.2-8.2
Positive Factor VIII Inhibitor Control: HEPES buffered (pH 6.2-8.2)
immunodepleted FVIII deficient plasma to which anti-human FVIII antibodies have been added.
Comparison to Predicate
Item	Predicate	New Device
Proprietary and Established Names	Factor VIII Antibody Screen,Immucor FVIII Antibody Screen Assay	CRYOCheck FVIII Inhibitor Kit
Manufacturer	Genetic Testing Institute, Inc.(original applicant)	Precision BioLogic
Similarities
Measurand	Antibodies to human Factor VIII	Factor VIII Inhibitor
Product Code	GGPTest, Qualitative and Quantitative Factor Deficiency	GGPTest, Qualitative andQuantitative Factor DeficiencyGGNPlasma, Coagulation Control
Regulation Section	21 CFR 864.7290Factor Deficiency Test	21 CFR 864.7290Factor Deficiency Test21 CFR 864.5425Multipurpose system for in vitrocoagulation studies
Classification	Class II	Class II
Panel	81 (Haematology)	81 (Haematology)
Intended Use	The Factor VIII Antibody Screen is aqualitative solid phase enzymelinked immunosorbent assay (ELISA)designed to detect IgG antibodiesreactive with recombinant humanVIII (FVIII) in human serum andplasma.	The CRYOcheck FVIII Inhibitor Kitis for clinical laboratory use inconjunction with a Factor VIIIactivity assay to enableperformance of a modifiedNijmegen-Bethesda assay using3.2% citrated human plasma. Itenables the determination of afunctional FVIII inhibitor titer toaid in the clinical management ofcongenital hemophilia A inindividuals aged 2 years or older.For in vitro diagnostic use.
Differences
Assay Type	ELISA(Qualitative)	Quantitative
Device Description	The Factor VIII Antibody Screenassay is an ELISA with a colorimetricendpoint. The assay kit is made of a96 welled microwell plate (in 1 X 8	The FVIII Inhibitor Kit is used inthe CDC modification of theNijmegen-Bethesda assay and
	strips), alkaline phosphataseconjugated goat antibody to humanimmunoglobulin G, p-nitrophenylphosphate substrate, positive andnegative control, Stop Solution,diluents, buffers, wash solutions,and plate sealers.	contains the followingcomponents:Imidazole Buffered PooledNormal Plasma (IB-PNP): Poolednormal plasma from a minimumof twenty donors with a factorVIII activity value of 95-113% andbuffered with imidazole to a pHof 7.3 – 7.5.Imidazole Buffered Bovine SerumAlbumin (IB-BSA): A 4% BSAsolution buffered with imidazoleto a pH of 7.3-7.5.Negative Factor VIII InhibitorControl: Pooled normal plasmafrom a minimum of five donorsbuffered with HEPES to a pH of6.2-8.2.Positive Factor VIII InhibitorControl: HEPES buffered (pH 6.2-8.2) immunodepleted FVIIIdeficient plasma to which anti-human FVIII antibodies havebeen added.
Methodology	Diluted patient sample is added tomicrowells coated withrecombinant FVIII, and antibody, ifpresent will bind. Unbound materialis washed away, and an alkalinephosphatase labeled anti-humanimmunoglobulin reagent (anti IgG)is added to the wells and incubated.Unbound anti-IgG is washed awayand then PNPP (p-nitrophenylphosphate) substrate is added.After a 30 minute incubationperiod, the reaction is stopped, andthe optical density of the color thatdevelops is measured at 405nm.	Diluted heat inactivated patientplasma is mixed with anexogenous source of FVIII (IB-PNP) and prepared alongside acontrol mix, consisting of adiluent (IB-BSA) and IB-PNP.Following a 2 hour incubation,the FVIII activity of the testplasma relative to the controlmix is determined and used tocalculate FVIII inhibitor activityexpressed as Bethesda units permilliliter.
Expression of results	Qualitative; results are reported aspositive or negative.	Quantitative; results areexpressed in Bethesda units permilliliter.

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Performance Summary:

All studies were performed using the cRYOcheck FVIII Inhibitor Kit in conjunction with the Siemens FVIII chromogenic assay (K884544, original applicant Baxter Healthcare Corporation) on Siemens BCS XP instrument(s) (K970431, original applicant Behring Diagnostics Inc).

Multi-Reagent Lot Precision

An internal precision study was performed using three (3) different lots of cRYocheck FVIII Inhibitor Kit by one operator on a Siemens BCS XP analyzer in accordance with CLSI EPO5-A3. The study quantified the kit's Positive and Negative Control as well as four plasma samples from congenital hemophilia A patients representing negative, low, mid and high levels of FVIII inhibitor. Each sample was measured with each product lot in duplicate, twice a day for 20 days for a total of 80 replicates per sample per lot. The results demonstrated a pooled precision of 8 % CV for the positive samples and 0.1 BU/mL SD for the negative sample as summarized below.

Lot 1
	Mean	Within-Run		Between- Run		Between-Day		Within-Lot
Sample	(BU/mL)	SD	%CV	SD	%CV	SD	%CV	SD	%CV
Kit Negative Control	0.0	0.0	189.7	0.0	178.9	0.0	156.4	0.1	304.1
Kit Positive Control	1.8	0.1	7.9	0.1	5.2	0.0	0.0	0.2	9.4
Negative Plasma Sample	0.3	0.1	22.2	0.0	10.1	0.0	0.0	0.1	24.4
Low Plasma Sample	1.4	0.1	7.9	0.0	0.0	0.0	2.2	0.1	8.2
Mid Plasma Sample	5.6	0.5	8.8	0.2	3.5	0.2	3.1	0.6	9.9
High Plasma Sample	9.6	0.6	6.1	0.2	2.0	0.2	2.0	0.6	6.7

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Lot 3
Sample	Mean(BU/mL)	Within-Run		Between- Run		Between-Day		Within-Lot
		SD	%CV	SD	%CV	SD	%CV	SD	%CV
Kit Negative Control	0.0	0.1	209.8	0.0	161.2	0.0	0.0	0.1	264.6
Kit Positive Control	1.5	0.1	8.4	0.1	4.2	0.1	5.5	0.2	10.9
Negative Plasma Sample	0.3	0.1	23.9	0.0	8.4	0.0	12.8	0.1	28.4
Low Plasma Sample	1.1	0.1	6.7	0.0	4.3	0.0	2.3	0.1	8.2
Mid Plasma Sample	5.3	0.3	6.0	0.2	3.8	0.1	1.3	0.4	7.2
High Plasma Sample	8.4	0.6	7.6	0.4	4.5	0.1	0.6	0.7	8.9

Aggregated Data (Lots 1, 2 and 3)

Sample	Mean(BU/mL)	Within-Laboratory
		SD	%CV
Negative Plasma Sample	0.3	0.1	29.0
Low Plasma Sample	1.2	0.1	8.2
Mid Plasma Sample	5.3	0.4	8.3
High Plasma Sample	8.6	0.7	8.4

Multi-Reagent Lot Site to Site Reproducibility

Reproducibility studies were conducted at three sites (one internal and two external) by three different operators on three different Siemens BCS XP analyzers using a single lot of cRYocheck FVIII Inhibitor Kit in accordance with CLSI EP05-A3. The study quantified the kit's Positive and Negative Control as well as four plasma samples from congenital hemophilia A patients representing negative, low, mid and high levels of FVIII inhibitor. Each sample was measured in triplicate, twice a day for 5 days for a total of 30 replicates per sample per site. The data across three sites demonstrated a pooled reproducibility of ≤16% CV for the positive samples and 0.1 BU/mL SD for the negative plasma sample as summarized below.

Pooled 3-Site Data
	Mean	Within-Run		Between- Run		Between-Day		Between-Site		Across-Site
Sample	(BU/mL)	SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
Kit Negative Control	0.0	0.1	399.4	0.0	138.3	0.0	0.0	0.0	0.0	0.1	422.7
Kit Positive Control	1.8	0.2	8.9	0.1	3.3	0.1	3.4	0.2	12.4	0.3	16.0
Negative Plasma Sample	0.3	0.1	26.9	0.0	0.0	0.0	9.0	0.0	2.2	0.1	28.4
Low Plasma Sample	1.4	0.1	10.2	0.0	0.0	0.0	2.9	0.0	0.0	0.1	10.6
Mid Plasma Sample	5.5	0.4	7.8	0.1	1.9	0.1	2.2	0.0	0.0	0.5	8.3
High Plasma Sample	9.6	0.7	7.8	0.4	4.0	0.3	3.4	0.9	9.2	1.3	13.2

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Linearity/Assay Reportable Range

A linearity study was conducted in accordance with CLSI EP06-A using one lot of cRYOcheck FVIII Inhibitor Kit. Factor VIII inhibitor positive plasma from a congenital hemophilia A patient was combined with congenital hemophilia A patient plasma containing no FVIII inhibitor to create twelve sample dilutions with estimated titers in the range of 0 to 100 BU/mL. The results support the linearity claim described below.

An expanded linearity study was also conducted using one lot of cRYOcheck FVIII Inhibitor Kit. A series of eighteen sample dilutions spanning a range of 0 to 1402.9 BU/mL was created by diluting a high titer contrived sample (FVIII inhibitor titer of 1402.9 BU/mL, comprised of FVIII monoclonal antibody in immunodepleted FVIII deficient plasma) with immunodepleted FVIII deficient plasma. The results support the linearity claim described below.

Linearity Range: 0.2 to 1402.9 BU/mL

Stability

Shelf Life Stability

A shelf life stability study was conducted in accordance with CLSI EP25-A. Three lots of cRYocheck FVIII Inhibitor Kit were stored at <-70°C (monitored condition -76 to -82°C) and tested at t=0 and regular intervals defined by the lot-specific pull schedule up to 37 months. At each timepoint, five replicates of the kit's Positive and Negative Control as well as four plasma samples from congenital hemophilia A patients representing negative, low, mid and high levels of FVIII inhibitor were quantified. The study has been completed up to 13 months and supports a 12-month shelf-life stability claim.

In-Use Stability

An in-use stability study was conducted in accordance with CLSI EP25-A. Three lots of cRYOcheck FVIII Inhibitor Kit were maintained at room temperature (18–25 °C) or in a refrigerator (2–8 °C) and used to quantify six replicates of the kit's Positive and Negative Control as three plasma samples from congenital hemophilia A patients representing negative, low and high levels of FVIII inhibitor at 0, 2, 4, 6 and 7 hours. The data support a 4-hour in-use stability of the product when maintained at room temperature or at 2-8 °C post thaw.

Detection Limit

The limit of blank (LoB) was determined following the CLSI EP17-A2 guideline by measuring four blank plasma samples obtained from normal healthy donors. Samples were measured in triplicate using three lots of cryocheck FVIII Inhibitor Kit over five days. The LoB was determined to be 0.1 BU/mL.

The limit of detection (LoD) was determined following the CLSI EP17-A2 guideline by measuring four low titer plasma samples obtained from congenital hemophilia A donors. Samples were measured in triplicate using three lots of cRYocheck FVIII Inhibitor Kit over five days. The LoD was determined to be 0.2 BU/mL.

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The limit of quantitation (LoQ) study was determined according to the CLSI EP17-A2 guideline. Aliquots of each low titer sample used in the LoD study were sent to an external laboratory for testing in three replicates on three different days to determine assigned values using a validated, laboratory-developed, CDC-modified Nijmegen-Bethesda assay. The LoQ was determined to be 0.2 BU/mL.

Interferences

Interference studies were conducted according to CLSI EP7-A2 using a single lot of cRYocheck FVIII Inhibitor Kit. Patient plasma samples were spiked with possible interferents and 10 replicates were tested alongside 10 replicates of the corresponding blank matrix control. The following substances showed no interference up to the concentrations indicated:

Possible Interferent	Concentration
Hemoglobin	≤ 500mg/dL
Bilirubin	≤ 500mg/dL
Intralipid	≤ 29mg/dL
vWF	≤ 20 µg/mL

Lupus anticoagulant autoantibodies may interfere with the quantification of low titer FVIII inhibitors.

Rheumatoid Factor at ≥ 82 IU/mL showed interference with the quantification of FVIII inhibitors.

Method Comparison Studies

A method comparison study was conducted at three sites (one internal and two external) according to CLSI EP09c to compare the accuracy of the cRYOcheck FVIII Inhibitor Kit relative to a comparator device. Aliquots of human plasma samples from individuals with congenital hemophilia A (N=210) were distributed across three sites and tested using a single lot of CRYOcheck FVIII Inhibitor Kit. A second aliquot of each sample was tested at a central reference laboratory using a chromogenic CDC-Modified Nijmegen-Bethesda Assay which has been implemented as a validated Laboratory-Developed Test on a Siemens BCS XP instrument. The data demonstrated positive agreement of 100% (95% Cl, 97-100%) and negative agreement of 99% (95% CI, 93-100%), as summarized below.

		cryocheck FVIII Inhibitor Kit results
		Positive	Negative	Total
Comparator deviceresults	Positive	133	0	133
	Negative	1	76	77
	Total	134	76	210

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Results were compared by Passing-Bablok regression analysis as well as Bland-Altman plots. Regression statistics show that the cRYOcheck FVIII Inhibitor Kit performed equivalently to the comparator method.

	N	Slope		Intercept		Pearson CorrelationCoefficient
		Value	95% CI	Value	95% CI
Site 1	68	1.543	1.404, 1.660	-0.224	-0.434, -0.127	0.977 ( $r^2$ =0.954)
Site 2	72	1.065	0.998, 1.190	-0.113	-0.138, -0.043	0.985 ( $r^2$ =0.969)
Site 3	70	1.373	1.225, 1.455	-0.075	-0.159, -0.023	0.980 ( $r^2$ =0.960)
Overall	210	1.341	1.265, 1.406	-0.145	-0.180, -0.070	0.970 ( $r^2$ =0.940)

Absolute predicted biases at medical decision levels are reported below.

Titer (BU/mL)	Predicted Bias (BU/mL)	Lower CI (%)	Upper CI (%)
0.6	-2.0	-4.8	0.8
1	-1.9	-4.6	0.9
5	-0.3	-3.0	2.5
10	1.7	-0.9	4.4

Sample Integrity

Sample integrity was assessed in two studies. In the first study, three test plasma samples were created by spiking anti-FVIII monoclonal antibody into immunodepleted Factor VIII deficient plasma representing negative, low and high levels of FVIII inhibitor and were quantified freshly prepared at t=0 and after 1) 25 hours storage at 2-8 °C, 2) freezing at -80°C and transfer to 2-8 °C for up to 25 hours, 3) freezing at -80°C and transfer to -20°C for up to 15 days, 4) freezing at -80°C, transfer to -20°C and up to three freeze/thaw cycles at -20°C and 5) freezing at -80°C and up to three freeze/thaw cycles at -80°C. The titers of immunodepleted FVIII-deficient plasma samples spiked with monoclonal anti-FVIII antibody measured consistently across each of these sample storage conditions, demonstrating robustness of FVIII measurements in fresh and frozen plasma samples including after multiple freeze/thaw cycles.

In the second study, a retrospective analysis was performed examining FVIII inhibitor titer results for four plasma samples from congenital hemophilia A patients representing negative, low, mid and high levels of FVIII inhibitor quantified using three lots of cryocheck FVIII Inhibitor Kit. The results demonstrated sample stability of 12 months when stored frozen at <-70°C.

Conclusion

The performance testing results demonstrate that the cRYocheck FVIII Inhibitor Kit is substantially equivalent to the predicate device, Immucor FVIII Antibody Screen Assay (K082205) and the comparator assay (validated Laboratory-Developed test), and that the assay is effective for its labeled intended use.

Regulation Number and Section

§ 864.7290 Factor deficiency test.

(a)
Identification. A factor deficiency test is a device used to diagnose specific coagulation defects, to monitor certain types of therapy, to detect coagulation inhibitors, and to detect a carrier state (a person carrying both a recessive gene for a coagulation factor deficiency such as hemophilia and the corresponding normal gene).(b)
Classification. Class II (performance standards).