CRYOcheck Chromogenic Factor IX is for clinical laboratory use in the quantitative determination of factor IX activity in 3.2% citrated human plasma. It is intended to be used in identifying factor IX deficiency and as an aid in the management of hemophilia B in individuals aged 2 years and older. For in vitro diagnostic use.

Device Description

CRYOcheck Chromogenic Factor IX is used for determination of FIX activity and contains the following four components, packaged in vials, and provided frozen to preserve the integrity of the components:
Reagent 1: Human FVIII, human FX, bovine FV and a fibrin polymerization inhibitor.
Reagent 2: Human FXIa, human FII, calcium chloride and phospholipids
Reagent 3: FXa Substrate containing EDTA and a thrombin inhibitor.
Diluent Buffer: Tris buffer solution containing 1% BSA and a heparin antagonist.

AI/ML Overview

The provided text describes the performance of the CRYOcheck Chromogenic Factor IX device. Here's a breakdown of the acceptance criteria and the study that proves the device meets these criteria, based on the information provided:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria with pass/fail thresholds for each performance characteristic. Instead, it presents study results and concludes that the device performed effectively and demonstrates substantial equivalence. However, we can infer some criteria from the performance data presented.

Performance Characteristic	Reported Device Performance (Implicit Acceptance)
Multi-Reagent Lot Precision	Within-Laboratory CV: Reference Control Normal (3.7%), Abnormal 1 (4.5%), Abnormal 2 (7.3%). Very Low FIX Plasma (14.5%), Low FIX Plasma (10.1%), High FIX Plasma (3.7%). These CVs are generally considered acceptable for diagnostic assays.
Multi-Reagent Lot Reproducibility	Across-Site CV: Reference Control Normal (5.6%), Abnormal 1 (6.6%), Abnormal 2 (8.6%). Very Low FIX Plasma (15.7%), Low FIX Plasma (13.0%), High FIX Plasma (6.0%). These CVs indicate good reproducibility across different sites and instruments.
Linearity	Linearity Range: 0 to 200% FIX activity. The study results are reported to "support the linearity claim."
Reference Interval	Reference Interval: 79 to 155% FIX activity. Established by calculating the non-parametric 95% confidence interval (2.5th to 97.5th percentiles) from 128 normal individuals.
Shelf-Life Stability	Shelf-Life Stability: At least 18 months when stored at ≤-70 ℃. The study was completed up to 19 months.
In-Use Stability	On-Board Stability: 24 hours on board the instrument. Refrigerated Stability: 48 hours at 2-8 °C. Refrozen Stability: One month at ≤-70 ℃ if stored on-board and refrozen within 4 hours, and used within eight hours of next thawing while kept on-board.
Detection Limit	Limit of Blank (LoB): 0.4% FIX activity. Limit of Detection (LoD): 0.5% FIX activity. Limit of Quantitation (LoQ): 0.5% FIX activity. These values indicate the assay's ability to detect and quantify low levels of FIX activity.
Interferences	No Interference: Hemoglobin (≤ 1000 mg/dL), Intraplipid (≤ 2000 mg/dL), Bilirubin (unconjugated ≤ 40 mg/dL), Bilirubin (conjugated ≤ 23 mg/dL), Unfractionated heparin (≤ 1.2 IU/mL), Low molecular weight heparin (≤ 1.5 IU/mL), Dabigatran (≤ 0.04 mg/L), Fondaparinux (≤ 0.26 mg/L), Lupus Anticoagulant (≤ 1.8 dRVVT ratio). Interference: Rivaroxaban and warfarin. This indicates the range of substances that do not affect the assay result.
Recovery of FIX Replacements	Mean Percent Recovery: AlphaNine SD (96%), Alprolix (116%), BeneFIX (93%), Ixinity (82%), Rebinyn (117%), Rixubis (102%). Overestimation: Idelvion (153%). The recoveries demonstrate the device's ability to evaluate the potency of most FIX concentrates.
Method Comparison	Pearson Correlation Coefficient: Ranging from 0.979 to 0.996 across sites, with an overall of 0.992 (r2=0.983). Passing-Bablok Regression: Slopes ranging from 1.05 to 1.21, intercepts from -11.76 to 2.44, and overall slope of 1.10 and intercept of 0.64. The study concludes that the device "performed equivalently to the comparator method."
Sample Integrity	Fresh Sample Stability: 4 hours at room temperature. Frozen Storage Stability: 3 months at ≤-70 °C, including up to two freeze-thaw cycles.
Overall Conclusion	The performance testing results demonstrate that CRYOcheck Chromogenic FIX is substantially equivalent to the predicate device and the comparator assay, and that the assay is effective for its labeled intended use.

2. Sample Size Used for the Test Set and Data Provenance

Multi-Reagent Lot Precision: 6 samples (1 normal reference control, 2 abnormal reference controls, 3 patient plasma samples).
Multi-Reagent Lot Site to Site Reproducibility: 6 samples (1 normal reference control, 2 abnormal reference controls, 3 patient plasma samples).
Linearity/Assay Reportable Range: 14 sample dilutions.
Reference Interval: 128 normal, ostensibly healthy individuals.
Shelf-Life Stability: Not specified, but likely involved multiple reference controls and patient plasma samples at each time point.
In-Use Stability: Not specified, but involved multiple reference controls and patient plasma samples.
Detection Limit (LoB): 4 blank plasma samples from individuals with severe congenital hemophilia B.
Detection Limit (LoD): 4 plasma samples with low FIX activity from congenital hemophilia B donors.
Detection Limit (LoQ): 4 plasma samples with low FIX activity from congenital hemophilia B donors.
Interferences: Not specified, but likely involved spiked plasma samples.
Recovery of FIX Replacements: Congenital FIX deficient plasma spiked with 7 FIX replacement therapies at 7 concentrations.
Method Comparison Studies: 368 human plasma samples. These samples were from normal ostensibly healthy individuals, patients with von Willebrand disease, patients with congenital and acquired hemophilia A and B, and patients on recombinant factor IX treatments.
Sample Integrity: 65 plasma samples.

Data Provenance: The document does not explicitly state the country of origin for the patient or donor samples. The studies involve both internal and external sites for reproducibility and method comparison. The mention of "congenital hemophilia B donors" and "patients with von Willebrand disease, and congenital and acquired hemophilia A and B" suggests the use of patient samples, implying a clinical or diagnostic context. The studies are retrospective in nature as they involve testing existing plasma samples.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

For the studies described, the "ground truth" for Factor IX activity is established by:

Reference controls: These are quality control materials with known or assigned values for FIX activity.
Patient plasma samples: Their FIX activity is determined either by the device itself (for precision and reproducibility) or by a "comparator method" or "validated laboratory developed chromogenic factor IX assay" (for method comparison and LoQ studies).
Spiked samples: For linearity, interference, and recovery studies, known amounts of FIX or interfering substances are added to samples to create a controlled "ground truth."
Congenital FIX deficient plasma: For LoB, LoD, and recovery studies, plasma from individuals with a known severe deficiency serves as a baseline.

The document does not mention specific "experts" in the context of radiologists or similar roles establishing ground truth through consensus. Instead, the ground truth is based on:

Established assay methods: The comparator method and the validated laboratory developed chromogenic factor IX assay are implicitly considered accurate for determining FIX activity. The qualifications of the personnel running these assays are not individually listed but are assumed to be trained laboratory professionals.
Reference materials: Reference controls have assigned values.

4. Adjudication Method for the Test Set

The concept of an "adjudication method" (like 2+1 or 3+1) is typically relevant for studies where human expert interpretation is compared, e.g., in medical image analysis. In this context of an in vitro diagnostic device for quantitative determination in plasma, such an adjudication method is not described or applicable. The "ground truth" is based on the results of the reference methods, reference materials, and defined sample preparations.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This type of study is not relevant for an in vitro diagnostic device like the CRYOcheck Chromogenic Factor IX, which directly measures a biomarker rather than assisting human readers in interpreting complex data like medical images. The device itself is the "reader" providing a quantitative result.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the studies presented are all standalone (algorithm only) performance assessments for the CRYOcheck Chromogenic Factor IX device. The device itself performs the quantitative determination of factor IX activity. The results are generated by the instrument (IL ACL TOP Series or TOP 50 Series Instruments) based on the reagents and the plasma sample, without human interpretative input in the output. Human operators are involved in running the tests, but not in interpreting the quantitative output in a way that typical human-in-the-loop AI studies would assess.

7. The Type of Ground Truth Used

The ground truth used in these studies includes:

Assigned values of reference controls: For precision and reproducibility.
Results from a "comparator device" or "validated laboratory developed chromogenic factor IX assay": For method comparison and limit of quantitation. These are considered gold standards for FIX activity measurement.
Known concentrations in spiked samples: For linearity, interference, and recovery studies.
Known characteristics of congenital FIX deficient plasma: For detection limits and recovery studies.
Plasma samples from "normal, ostensibly healthy individuals": For establishing reference intervals.

This primarily falls under the category of established assay methods and reference materials.

8. The Sample Size for the Training Set

The document does not mention a "training set" in the context of machine learning or AI. The CRYOcheck Chromogenic Factor IX is an in vitro diagnostic assay, not an AI/ML algorithm that requires training data. Its performance is based on chemical and enzymatic reactions, and its parameters are established through conventional analytical validation studies like those described.

9. How the Ground Truth for the Training Set Was Established

As there is no "training set" in the AI/ML sense for this device, this question is not applicable. The device's operational parameters and performance are intrinsically defined by its design, chemical components, and the analytical validation tests presented.

Summary

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December 23, 2022

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

Re: K214002

Trade/Device Name: Cyrocheck Chromogenic Factor IX Regulation Number: 21 CFR 864.7290 Regulation Name: Factor Deficiency Test Regulatory Class: Class II Product Code: GGP Dated: December 20, 2021 Received: December 21, 2021

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 statutes and regulations administered by other Federal agencies. You must comply with all the Act's

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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/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) 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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

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Min Wu Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration

Indications for Use

510(k) Number (if known) K214002

Device Name

CRYOcheck™ Chromogenic Factor IX

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™ Chromogenic Factor IX

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 K214002

Submitter's Information	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	20 December 2022
Device Trade Name	CRYOCheck™ Chromogenic Factor IX
RegulatoryInformation	Regulation Number andDescription	21 CFR 864.7290Factor Deficiency Test
	Classification	Class II
	Product Code	GGP; Test, Qualitative and QuantitativeFactor Deficiency; 21 CFR 864.7290
	Classification Panel	Hematology
Predicate Device	HemosIL Factor IX Deficient Plasma (K031829)
Indication for Use/IntendedUse	CRYOCheck Chromogenic Factor IX is for clinical laboratory use in thequantitative determination of factor IX activity in 3.2% citrated humanplasma. It is intended to be used in identifying factor IX deficiency and asan aid in the management of hemophilia B in individuals aged 2 yearsand older. For in vitro diagnostic use.
Device Description	CRYOCheck Chromogenic Factor IX is used for determination of FIXactivity and contains the following four components, packaged in vials,and provided frozen to preserve the integrity of the components:Reagent 1: Human FVIII, human FX, bovine FV and a fibrinpolymerization inhibitor.Reagent 2: Human FXIa, human FII, calcium chloride and phospholipidsReagent 3: FXa Substrate containing EDTA and a thrombin inhibitor.Diluent Buffer: Tris buffer solution containing 1% BSA and a heparinantagonist.
	Comparison to Predicate
Item	Predicate	New Device
Proprietary andEstablished Names	HemosIL Factor IX DeficientPlasma	CRYOCheck Chromogenic Factor IX
Manufacturer	Instrumentation Laboratory	Precision BioLogic
Similarities
Measurand	Human Factor IX	Human Factor IX
Product Code	Classification Product Code:GJT; Factor deficiency testSubsequent Product Code: GGPTest, Qualitative and QuantitativeFactor Deficiency	GGPTest, Qualitative and QuantitativeFactor Deficiency

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Item	Predicate	New Device
Regulation Section	21 CFR 864.7290	21 CFR 864.7290
	Factor Deficiency Test	Factor Deficiency Test
Classification	Class II	Class II
Panel	81 (Haematology)	81 (Haematology)
Intended Use	Human plasma immunodepletedof factor IX for the quantitativedetermination of factor IX activityin citrated plasma, based onactivated partial thromboplastintime (APTT) assay, on ILCoagulation Systems.	CRYOcheck Chromogenic Factor IXis for clinical laboratory use in thequantitative determination of factorIX activity in 3.2% citrated humanplasma. It is intended to be used inidentifying factor IX deficiency andas an aid in the management ofhemophilia B in individuals aged 2years and older. For in vitrodiagnostic use.
Assay Type	Quantitative (clot-based	Quantitative (chromogenic
	measurement of FIX)	measurement of FIX)
Expression of results	Quantitative; results areexpressed as percent activityinterpreted relative to acalibrationcurve.	Quantitative; results are expressedas percent activity interpretedrelativeto a calibration curve.
Instrument(s)	IL Coagulation Systems	ACL TOP Family/
		ACL TOP Family 50 Series
	Differences
Device Description	The Factor IX deficient plasma kitconsists of: Factor IX deficientplasma (Cat. No. 0020011910):10 x 1 mL vials of lyophilizedhuman plasma that has beenartificially depleted of factor IXcontaining buffer and stabilizers.The residual factor IX activity isless than or equal to 1% whereasall other coagulation factors havenormal levels.	CRYOcheck Chromogenic Factor IX isused for determination of FIX activity andcontains the following four components,packaged in vials and provided frozen topreservethe integrity of the components:Reagent 1: Human FVIII, human FX,bovine FV and a fibrin polymerizationinhibitor.Reagent 2: Human FXIa, human FII,calcium chloride and phospholipids.Reagent 3: FXa Substrate containingEDTA and a thrombin inhibitor.Diluent Buffer: Tris buffer solutioncontaining 1% BSA and a heparinantagonist
Methodology	Factor IX activity in a patient'splasma is determined byperforming a modified activatedpartial thromboplastin time test(APTT). Patient plasma is dilutedand added to a plasma deficient infactor IX. Correction of the clottingtime of the deficient plasma isproportional to the concentration(% activity) of that factor in thepatient plasma, interpolated from acalibration curve.	FIX activity is determined in achromogenic method, in which humanFIX is activated by human FXIa andwhere formed FIXa activates human FXin the presence of human FVIII, calciumions and phospholipid. Similar to in vivoconditions, FVIII is activated by thrombinwhich is generated during the incubation.The amountof FXa formed is related tothe FIX activity and is determined fromthe hydrolysis of a chromogenic FXasubstrate. The color produced by therelease of pNA is measuredspectrophotometrically at 405 nm and isproportional to the factor IX in thesample.

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Performance Summary:

All studies were performed using CRYOcheck Chromogenic Factor IX on Instrumentation Laboratories' ACL TOP Series or TOP 50 Series Instruments; the specific instrument(s) used for each study are indicated in the summary reports below.

Multi-Reagent Lot Precision

An internal precision study was performed using three (3) lots of CRYOcheck Chromogenic Factor IX by two operators on an IL ACL TOP 700 CTS analyzer (K160276) in accordance with CLSI EP05-A3. The study quantified one normal and two abnormal reference controls and three patient plasma samples representing very low, low and high levels of FIX activity. 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.

Aggregated Data (Lots 1, 2 and 3)
Sample	Mean FIX (%)	Within-Laboratory
		SD	%CV
CRYOcheck Reference Control Normal	114.9	4.2	3.7
CRYOcheck Abnormal 1 Reference Control	39.3	1.8	4.5
CRYOcheck Abnormal 2 Reference Control	10.4	0.8	7.3
Very Low FIX Plasma Sample	1.2	0.2	14.5
Low FIX Plasma Sample	6.1	0.6	10.1
High FIX Plasma Sample	174.0	6.4	3.7

Multi-Reagent Lot Site to Site Reproducibility

Reproducibility studies were conducted at three sites (one internal and two external) by two operators per site on IL ACL TOP 700 CTS (K160276), IL ACL TOP 700 (K160276) and IL ACL TOP 750 CTS (K150877) analyzers using three lots of cRYOcheck Chromogenic Factor IX in accordance with CLSI EP05-A3. The study quantified one normal and two abnormal reference controls and three patient plasma samples representing very low, low and high levels of FIX activity. Each sample was measured in triplicate, twice a day for 5 days at each site.

Pooled 3-Site Data
Sample	Mean (%)	Within-Run		Between- Run		Between-Day		Between-Site		Across-Site
		SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
Reference Control Normal	113.1	5.0	4.5	0.9	0.8	1.1	1.0	1.9	1.7	6.3	5.6
Abnormal 1 Reference Control	38.4	1.8	4.8	0.2	0.6	0.1	0.3	1.3	3.4	2.5	6.6
Abnormal 2 Reference Control	10.8	0.8	7.5	0.2	1.7	0.0	0.0	0.2	2.0	0.9	8.6
Very Low FIX Plasma Sample	1.2	0.1	6.9	0.0	1.7	0.0	0.0	0.0	0.0	0.2	15.7
Low FIX Plasma Sample	6.3	0.5	7.9	0.1	1.2	0.1	2.1	0.2	2.4	0.8	13.0
High FIX Plasma Sample	168.5	7.6	4.5	0.0	0.0	1.3	0.8	4.4	2.6	10.0	6.0

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Linearity/Assay Reportable Range

A linearity study was conducted in accordance with CLSI EP06-2™ Ed using three lots of cRYocheck Chromogenic Factor IX on an IL ACL TOP 700 CTS instrument (K160276). A high FIX (230%) plasma was combined with conqenital FIX deficient plasma (0%) to create fourteen sample dilutions with estimated FIX activities in the range of 0 to 230% FIX. Each level was tested in quadruplicate. The results support the linearity claim described below.

Linearity Range: 0 to 200% FIX activity

Reference Interval

A reference interval study was conducted by two operators on IL ACL TOP 700 CTS and IL ACL TOP CTS instruments (K160276) in accordance with CLSI EP28-A3c using three lots of CRYocheck Chromogenic Factor IX and citrated plasma samples from 128 normal, ostensibly individuals. The reference interval was established by calculating the non-parametric 95% confidence interval (2.5th to 97.5th percentiles).

Reference Interval: 79 to 155% FIX activity

Stability

Shelf-Life Stability

A shelf-life stability study was conducted in accordance with CLSI EP25-A using an IL ACL TOP 700 CTS instrument (K160276). At each timepoint, five replicates of one normal and two abnormal reference controls and two patient plasma samples representing very low and high levels of FIX activity levels were quantified. The study has been completed up to 19 months and supports a shelf-life stability claim of at least 18 months when the product is stored at ≤-70 ℃.

In-Use Stability

An in-use stability study was conducted in accordance with CLSI EP25-A using an IL ACL TOP 700 CTS instrument (K160276). Each lot was used to quantify five replicates of one normal and two abnormal reference controls and two patient plasma samples representing very low and low levels of FIX activity levels from each storage condition at defined timepoints. The data support a stability claim of 24 hours on board the instrument and 48 hours at 2-8 °C.

Three lots of cRYocheck Chromogenic Factor IX were maintained on board an analyzer for 4 hours, then subsequently refrozen at ≤-70 ℃ for up to 3 months. Each lot was used to quantify five replicates of one normal and two abnormal reference controls and two patient plasma samples representing very low and low levels of FIX activity levels at defined timepoints. The data support a stability claim of one month refrozen storage at ≤-70 ℃ if the product is stored on-board and refrozen within 4 hours of the initial thaw. The refrozen product must be used within eight hours of next thawing while kept on-board the analyzer.

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Detection Limit

The limit of blank (LoB) was determined in accordance with CLSI EP17-A2 by measuring four blank plasma samples obtained from individuals with severe congenital hemophilia B. Samples were measured in triplicate on an IL ACL TOP 700 CTS instrument (K160276) using three lots of cRYocheck Chromogenic Factor IX over five days. The LoB was determined to be 0.4% FIX activity.

The limit of detection (LoD) was determined in accordance with CLSI EP17-A2 by measuring four plasma samples with low FIX activity obtained from congenital hemophilia B donors. Samples were measured in triplicate on an IL ACL TOP CTS instrument (K160276) using three lots of CRYOcheck Chromogenic Factor IX over five days. The LoD was determined to be 0.5%FIX activity.

The limit of quantitation (LoQ) was determined in accordance with CLSI EP17-A2. Aliquots of four plasma samples with low FIX activity obtained from congenital hemophilia B donors were sent to an external laboratory for three replicates on five different days on an IL ACL TOP 700 instrument (K160276) to determine assigned values using a validated laboratory developed chromogenic factor IX assay. The LoQ was determined to be 0.5% FIX activity.

Interferences

Interference studies were conducted according to CLSI EP07-A3 using a single lot of CRYocheck Chromogenic Factor IX on an IL ACL TOP 700 CTS instrument (K160276). 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	≤ 1000 mg/dL
Intraplipid	≤ 2000 mg/dL
Bilirubin (unconjugated)	≤ 40 mg/dL
Bilirubin (conjugated)	≤ 23 mg/dL
Unfractionated heparin	≤ 1.2 IU/mL
Low molecular weight heparin	≤ 1.5 IU/mL
Dabigatran	≤ 0.04 mg/L
Fondaparinux	≤ 0.26 mg/L
Lupus Anticoagulant	≤ 1.8 dRVVT ratio

Rivaroxaban and warfarin interfered with the quantification of FIX activity.

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Recovery of FIX Replacements

A recovery study was conducted using a single lot of CRYOcheck Chromogenic Factor IX on an IL ACL TOP 700 CTS instrument (K160276). Congenital FIX deficient plasma was spiked with seven FIX replacement therapies at seven concentrations and percent recovery was determined. CRYocheck Chromogenic Factor IX accurately evaluated the potency of FIX concentrates including AlphaNine® SD, Alprolix®, BeneFIX®, Ixinity, Rebinyn® and Rixubis at concentrations ranging from 0.05 to 1.0 IU/mL. There was an overestimation of Idelvion*.

Product	Mean Percent Recovery (%)
AlphaNine SD	96
Alprolix	116
BeneFIX	93
Ixinity	82
Rebinyn	117
Rixubis	102
Idelvion*	153

Per the manufacturer's recommendations, a one stage clotting assay is recommended for measurement of Idelvion and results may vary based on the aPTT reagent in use.

Method Comparison Studies

A method comparison study was conducted at four sites (one internal and three external) according to CLSI EP09c to compare the accuracy of CRYOcheck Chromogenic Factor IX relative to a comparator device. Three hundred and sixty eight human plasma samples from normal ostensibly healthy individuals, from patients with von Willebrand disease, from patients with congenital and acquired hemophilia A and B and patients on recombinant factor IX treatments were distributed across four sites and tested for FIX activity using a single lot of CRYOcheck Chromogenic Factor IX on IL ACL TOP 700 CTS (K160276), IL ACL TOP 700 (K160276) and IL ACL TOP 750 (K150877) analyzers. A second aliquot of each sample was tested at two central reference laboratories using a validated laboratory developed chromogenic factor IX assay on an IL ACL TOP 700 instrument (K160276).

Results were compared by Passing-Bablok regression statistics show that CRYOcheck Chromogenic Factor IX performed equivalently to the comparator method.

	N	Slope		Intercept		Pearson CorrelationCoefficient
		Value	95% CI	Value	95% CI
Site 1	108	1.11	1.08, 1.13	-0.01	-0.12, 0.17	0.996 (r2=0.991)
Site 2	112	1.17	1.13, 1.20	1.72	1.16, 2.38	0.995 (r2=0.990)
Site 3	112	1.21	1.15, 1.27	-11.76	-17.85, -7.26	0.979 (r2=0.959)
Site 4	36	1.05	1.02, 1.12	2.44	0.63, 3.61	0.993 (r2=0.987)
Overall	368	1.10	1.08, 1.12	0.64	0.20, 1.34	0.992 (r2=0.983)

Absolute predicted biases at medical decision levels are reported below.

FIX activity (%)	Predicted Bias (%)	Lower CI (%)	Upper CI (%)
1	0.74	0.32	1.40
5	1.14	0.75	1.76
50	5.66	5.09	6.40
100	10.68	9.38	12.07

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Sample Integrity

A sample integrity study was conducted at two external sites to assess the stability of fresh plasma samples at room temperature, when stored frozen at ≤-70 °C and after up to two freeze thaw cycles. The FIX activity of sixty-five plasma samples was measured using two lots of CRYOcheck Chromogenic Factor IX on IL ACL TOP 300 and IL ACL TOP 700 (K160276) analyzers. Results were compared using Passing Bablok regression analysis and support a fresh sample stability claim of 4 hours at room temperature and a frozen storage claim of 3 months at ≤-70 °C, including up to two freeze thaw cycles.

Conclusion

The performance testing results demonstrate that CRYOcheck Chromogenic FIX is substantially equivalent to the predicate device, HemosIL Factor IX Deficient Plasma (K031829) and the comparator assay (validated laboratory developed chromogenic FIX assay), 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).