CRYOcheck Chromogenic Factor IX

K214002 · Precision Biologic, Inc. · GGP · Dec 23, 2022 · Hematology

Device Facts

• Record ID: K214002
• Device Name: CRYOcheck Chromogenic Factor IX
• Applicant: Precision Biologic, Inc.
• Product Code: GGP · Hematology
• Decision Date: Dec 23, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7290
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

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 Story

CRYOcheck Chromogenic Factor IX is an in vitro diagnostic reagent kit used for the quantitative determination of Factor IX (FIX) activity in human plasma. The device is used in clinical laboratories on automated coagulation analyzers (ACL TOP family). The assay utilizes a chromogenic method: human FIX in the sample is activated by human FXIa; the resulting FIXa activates human FX in the presence of human FVIII, calcium ions, and phospholipids. The amount of FXa generated is proportional to the FIX activity in the sample and is determined by the hydrolysis of a chromogenic FXa substrate, measured spectrophotometrically at 405 nm. The output is expressed as percent activity relative to a calibration curve. This information aids clinicians in diagnosing FIX deficiency and managing hemophilia B. The device is provided frozen to preserve component integrity.

Clinical Evidence

Bench testing only. Performance evaluated on ACL TOP series analyzers. Precision (within-lab %CV 3.7-14.5%) and reproducibility (across-site %CV 5.6-15.7%) established. Linearity range 0-200% FIX activity. LoB/LoD/LoQ determined to be 0.4%, 0.5%, and 0.5% respectively. Method comparison (n=368) against a validated laboratory-developed chromogenic assay showed strong correlation (Pearson r=0.992) and equivalent performance. Interference studies identified Rivaroxaban and warfarin as interfering substances.

Technological Characteristics

Chromogenic assay; reagents include human FVIII, FX, FXIa, FII, bovine FV, fibrin polymerization inhibitor, phospholipids, and chromogenic FXa substrate. Form factor: Frozen liquid reagents. Connectivity: Integrated with IL ACL TOP/ACL TOP Family 50 Series analyzers. Measurement: Spectrophotometric at 405 nm. Standards: CLSI EP05-A3, EP06, EP07, EP17-A2, EP25-A, EP28-A3c.

Indications for Use

Indicated for quantitative determination of factor IX activity in 3.2% citrated human plasma to identify factor IX deficiency and aid in management of hemophilia B in individuals aged 2 years and older.

Predicate Devices

• HemosIL Factor IX Deficient Plasma (K031829)

Submission Summary (Full Text)

{0}

FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K214002

B Applicant

Precision BioLogic Inc.

C Proprietary and Established Names

CRYOcheck Chromogenic Factor IX

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| GGP | Class II | 21 CFR 864.7290 - Factor Deficiency Test | HE - Hematology |

## II Submission/Device Overview:

A Purpose for Submission:

Clearance of a new device

B Measurand:

Factor IX activity (%)

C Type of Test:

Quantitative chromogenic assay

## III Intended Use/Indications for Use:

A Intended Use(s):

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

{1}

See Indications for Use below.

## B Indication(s) for Use:

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.

## C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

## D Special Instrument Requirements:

Instrument Laboratory (IL) ACL TOP (K160276) and ACL TOP Family 50 Series (K150877)

## IV Device/System Characteristics:

### A 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 Name | Format (per vial set; each box contains 4 vial sets | Description |
| --- | --- | --- |
| Reagent 1 | 1 x 0.75 mL Frozen | Human FVIII, human FX, bovine FV and a fibrin polymerization inhibitor |
| Reagent 2 | 1 x 2.3 mL Frozen | Human FXIa, human FII, calcium chloride and phospholipids |
| Reagent 3 | 1 x 1.0 mL Frozen | FXa Substrate containing EDTA and thrombin inhibitor |
| Diluent Buffer | 1x 10.0 mL Frozen | Tris buffer solution containing 1% BSA and a heparin antagonist |

### B Principle of Operation:

CRYOcheck Chromogenic Factor IX is a chromogenic factor IX assay. In this assay, FIX activity is determined in a chromogenic method, in which human FIX is activated by human FXIa and where formed FIXa activates human FX in the presence of human FVIII, calcium ions and phospholipid. Similar to in vivo conditions, FVIII is activated by thrombin which is generated during the incubation. The amount of FXa formed is related to the FIX activity and is determined from the hydrolysis of a chromogenic FXa substrate. The color produced by the release of p-Nitroanilide (pNA) is measured spectrophotometrically at 405 nm and is proportional to the factor IX in the sample. FIX results are reported in percent activity where 100% FIX activity is equivalent to 1.0 IU/mL.

### V Substantial Equivalence Information:

K214002 - Page 2 of 10

{2}

A Predicate Device Name(s):

HemosIL Factor IX Deficient Plasma

B Predicate 510(k) Number(s):

K031829

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K214002 | K031829 |
| --- | --- | --- |
| Device Trade Name | CRYOcheck
Chromogenic Factor IX | HemosIL Factor IX
Deficient Plasma |
| General Device Characteristic Similarities | | |
| Intended Use/Indications for Use | 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 | Human plasma immunodepleted of factor IX for the quantitative determination of factor IX activity in citrated plasma, based on activated partial thromboplastin time (APTT) assay, on IL Coagulation Systems. |
| Classification | Class II | Same |
| Regulation Section | 21 CFR 864.7290
Factor Deficiency Test | Same |
| Type of Test | Quantitative | Same |
| Measurand | Human Factor IX activity | Same |
| Expression of Results | Quantitative; Results are expressed as percent activity interpreted relative to a calibration curve. | Same |
| Instrument | ACL TOP/ACL TOP Family 50 Series | ACL instruments/ACL TOP Family |
| General Device Characteristic Differences | | |
| Device Description | CRYOcheck
Chromogenic Factor IX
is used for | The Factor IX deficient plasma kit consists of: Factor IX deficient |

K214002 - Page 3 of 10

{3}

K214002 - Page 4 of 10
| | 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 | plasma (Cat. No. 0020011910): 10 x 1 mL vials of lyophilized human plasma that has been artificially depleted of factor IX containing buffer and stabilizers. The residual factor IX activity is less than or equal to 1% whereas all other coagulation factors have normal levels |
| --- | --- | --- |
| Methodology | FIX activity is determined in a chromogenic method, in which human FIX is activated by human FXIa and where formed FIXa activates human FX in the presence of human FVIII, calcium ions and phospholipid. Similar to in vivo conditions, FVIII is activated by thrombin which is generated during the incubation. The amount of FXa formed is related to the FIX activity and is determined from the hydrolysis of a chromogenic FXa substrate. The color produced by the release of pNA is measured spectrophotometrically | Factor IX activity in a patient’s plasma is determined by performing a modified activated partial thromboplastin time test (APTT). Patient plasma is diluted and added to a plasma deficient in factor IX. Correction of the clotting time is proportional to the concentration (% activity) of that factor in the patient plasma, interpolated from a calibration curve. |

{4}

| | at 405 nm and is proportional to the factor IX in the sample. | |
| --- | --- | --- |
| Calibrator and Control Plasma | CRYOcheck Reference Control Normal, CRYOcheck Abnormal 1 and CRYOcheck Abnormal 2 Reference Control | IL Coagulation Normal and Abnormal control |
| Storage | 18 months at ≤-70 °C | 3 weeks at -20°C |
| Assay Reportable Range | 0.5–200% FIX activity | 0.5–150% |
| Limit of Detection | LoB – 0.4% FIX activity
LoD – 0.5% FIX activity
LoQ – 0.5% FIX activity | NA |

VI Standards/Guidance Documents Referenced:

CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition

CLSI EP06, Evaluation of Linearity of Quantitative Measurement Procedures, 2nd Edition

CLSI EP07, Interference Testing in Clinical Chemistry, 3rd 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

CLSI EP28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility

a. Single Site Precision

This study was conducted at the internal site over 20 days with two runs per day and two replicates per run, for a total of 80 determinations per sample per reagent lot. Three lots of CRYOcheck Chromogenic Factor IX were tested with six panel members on a single IL ACL TOP 700 CTS by two operators alternating testing days. Test samples included one normal and two abnormal reference controls, as well as three patient plasma samples representing very low, low, and high levels of FIX activity. Precision estimates were calculated for each

K214002 - Page 5 of 10

{5}

of the following variance components: within-run, between-run, between-day, between-lot and within-laboratory precision. The results for within-run and total imprecision for all lots combined are provided in the summary table below.

| Sample | N | Mean (%) | Within-Run | | Between-Run | | Between-Day | | Between-lot | | Within-Laboratory | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Normal reference control | 240 | 114.9 | 3.9 | 3.4 | 0.2 | 0.2 | 1.6 | 1.4 | 1.1 | 0.9 | 4.2 | 3.7 |
| Abnormal 1 reference control | 240 | 39.3 | 1.3 | 3.4 | 0.5 | 1.2 | 0.4 | 1.1 | 1.1 | 2.8 | 1.8 | 4.5 |
| Abnormal 2 reference control | 240 | 10.4 | 0.7 | 6.3 | 0.2 | 1.6 | 0.2 | 2.0 | 0.3 | 3.3 | 0.8 | 7.3 |
| Plasma sample 1 | 240 | 1.2 | 0.1 | 9.8 | 0.0 | 0.9 | 0.0 | 2.4 | 0.1 | 10.3 | 0.2 | 14.5 |
| Plasma sample 2 | 240 | 6.1 | 0.5 | 7.9 | 0.0 | 0.0 | 0.2 | 2.8 | 0.4 | 6.3 | 0.6 | 10.1 |
| Plasma sample 3 | 240 | 174.0 | 5.4 | 3.1 | 3.3 | 1.9 | 0.8 | 0.5 | 0.4 | 0.2 | 6.4 | 3.7 |

# b. Multi-Site Reproducibility

This study was conducted at three testing sites over five days with two runs per day and three replicates per run for a total of 90 determinations. The study design included three different representative instrument models (one instrument per site). There were two operators at each site alternating test days. The study quantified three reagent lots testing one normal and two abnormal reference controls as well as three patient plasma samples representing very low, low, and high levels of FIX activity. Precision estimates were calculated for each of the following variance components: within-run, between-run, between-day, between-site and reproducibility. The results for overall reproducibility for all the lots combined are provided in the summary table below.

| Sample | N | Mean (%) | Within-Run | | Between-Run | | Between-Day | | Between-Site | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Normal reference control | 270 | 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 | 270 | 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 | 270 | 10.8 | 0.8 | 7.5 | 0.2 | 1.7 | 0.0 | 0.0 | 0.2 | 2.0 | 0.9 | 8.6 |
| Plasma sample 1 | 270 | 1.2 | 0.1 | 6.9 | 0.0 | 1.7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.2 | 15.7 |
| Plasma sample 2 | 270 | 6.3 | 0.5 | 7.9 | 0.1 | 1.2 | 0.1 | 2.1 | 0.2 | 2.4 | 0.8 | 13.0 |
| Plasma sample 3 | 270 | 168.5 | 7.6 | 4.5 | 0.0 | 0.0 | 1.3 | 0.8 | 4.4 | 2.6 | 10.0 | 6.0 |

# 2. Linearity:

The linearity studies were performed following the CLSI EP06 $2^{\mathrm{nd}}$ Ed guideline using three reagent lots at one site on a single IL ACL TOP 700 CTS instrument. Fourteen sample dilutions were prepared using a high FIX plasma ( $\sim 230\%$ ) with congenital FIX deficient plasma ( $\sim 0\%$ ) to create sample dilutions ranging from $0 - 230\%$ FIX activity. Each dilution was tested in four replicates. The linear range for CRYOcheck Chromogenic Factor FIX assay was determined to be $0 - 200\%$ .

# 3. Analytical Specificity/Interference:

Interference study was conducted in accordance with the CLSI EP07-A3 guideline. A single lot of CRYOcheck Chromogenic Factor IX was used on an IL ACL TOP 700 CTS instrument.

K214002 - Page 6 of 10

{6}

Potentially interfering endogenous and exogenous substances were spiked into the control samples (one normal and one abnormal reference control plasma). Ten replicates were evaluated for each spiked plasma or matrix-blank plasma. The maximum concentration of each interferent was determined per CLSI EP37 guideline. None of the substances in the following table (endogenous or exogenous) were found to lead to clinically significant interference (up to the concentrations indicated in the table below).

| Interferent | Concentration |
| --- | --- |
| Unconjugated Bilirubin | 40 mg/dL |
| Conjugated Bilirubin | 23 mg/dL |
| Hemoglobin | 1000 mg/dL |
| Intralipid | 2000 mg/dL |
| Dabigatran | 0.04 mg/L |
| Fondaparinux | 0.26 mg/L |
| Low Molecular Weight Heparin (LMWH) | 1.5 IU/mL |
| Unfractionated Heparin | 1.2 IU/mL |
| Lupus Anticoagulant | dRVVT ratio = 1.8 |

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

Traceability

Three levels of CRYOcheck Reference controls are prepared from human citrated plasma with three different concentrations of hemostatic parameters. CRYOcheck Reference Control Normal (K013708) was normal human citrated plasma while CRYOcheck Abnormal 1 Reference Control (K952624) and CRYOcheck Abnormal 2 Reference Control (K952624) were the citrated human plasma with hemostatic parameter values in the borderline pathological range 30% to 44% and 9% to 17%, respectively. Calibrator value assignments are traceable to the WHO International Standard for Factor IX (4th IS Factors II, VII, IX, X, Plasma, 09/172).

Sample Stability

Sample stability studies were performed to support the recommended storage and handling instructions found in the device labeling. Citrated plasma samples were tested after storage in the following temperature ranges, room temperature (18–22°C) and below -70°C. The study was performed using two reagent lots of CRYOcheck Chromogenic Factor IX on IL ACL TOP 300 and IL ACL TOP 700 analyzers with five replicate measurements at each time point for each sample. The study data demonstrate that citrated plasma samples are stable for four hours when stored at room temperature (18–22°C), three months when stored at <-70°C, including up to two freeze thaw cycles.

Shelf-life Stability

The shelf-life stability study was performed to determine the shelf-life stability of CRYOcheck Chromogenic Factor IX when stored at ≤-70 °C. The study was conducted with five plasma samples (one normal, two abnormal reference controls and two patient plasmas)

K214002 - Page 7 of 10

{7}

using three lots of CRYOcheck Chromogenic Factor IX on an IL ACL TOP 700 CTS analyzer. Samples were thawed from ≤-70 °C storage and tested in five replicates for FIX activity at the following timepoints: 0, 6, 7, 12, 13, 18, 19, 24, 25, 36 and 37 months. The study is ongoing and current data supports the shelf-life stability claim of 18 months when the product is stored at ≤-70 °C.

## In Use Stability

The in-use stability studies were performed to characterize in-use stability of CRYOcheck Chromogenic Factor IX assay when maintained at three different in-use conditions: on-board ACL TOP analyzer (12–18°C), in a refrigerator (2–8°C) and refrozen at ≤-70°C after maintaining on board for 3 hours. Five plasma samples (one normal, two abnormal reference controls and two patient plasmas) were tested by one operator on one IL ACL TOP 700 CTS analyzer. Samples were tested in five replicates by using three lots of CRYOcheck Chromogenic Factor IX at each storage condition at each timepoint. The study data supports the in-use stability claim of 24-hours when the product is stored on-board (12–18°C), 48 hours at 2–8°C and 1 month stored at ≤-70°C if the product is stored on board and refrozen within 4 hours of the initial thaw. The refrozen product must be used within 8 hours of next thawing while kept on-board.

## 5. Detection Limit:

The limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ) for test system were determined following the CLSI EP17-A2 guideline. Each study design included three reagent lots and one representative instrument model (IL ACL TOP 700 CTS).

The LoB was determined using four blank plasma samples obtained from individuals with severe congenital hemophilia B. Each sample was tested in triplicate using three lots of CRYOcheck Chromogenic Factor IX over five days on an IL ACL TOP instrument for N=15 determinations per reagent lot. The LoB was determined to be 0.4% FIX activity.

The LoD was determined using four plasma samples with low FIX activity obtained from individuals with congenital hemophilia B donors. Each sample was tested in triplicate using three lots of CRYOcheck Chromogenic Factor IX over five days on an IL ACL TOP instrument for N=15 determinations per reagent lot. The LoD was determined to be 0.5% FIX activity.

The LoQ was determined using four plasma samples with low FIX activity obtained from congenital hemophilia B donors. Samples were measured in triplicate using three lots of CRYOcheck Chromogenic Factor IX over five days on an IL ACL TOP 700 instrument. The samples were also measured in triplicate using a validated laboratory-developed chromogenic factor IX assay (ROX Factor IX) over five days to determine the assigned values. The LoQ was determined to be 0.5% FIX activity.

## 6. Assay Cut-Off:

Not Applicable

## 7. Recovery of FIX Replacement Therapies

K214002 - Page 8 of 10

{8}

The recovery of replacement therapy study was conducted to evaluate the recovery of FIX activity of seven FIX replacement therapies with one lot of CRYOcheck Chromogenic Factor IX. A congenital FIX deficiency plasma was used to prepare seven concentration levels for each FIX replacement therapy (1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 IU/mL). Ten replicates of each level of each product were tested on an IL ACL TOP 700 CTS analyzer. The FIX percent recovery was determined from the measured versus expected FIX activity (%) of each product at each level. CRYOcheck Chromogenic Factor IX recovered FIX activity levels in plasma containing AlphaNine, Alprolix, BeneFIX, Ixinity, Rebinyn and Rixubiz at concentrations ranging from 0.05 to 1.0 IU/mL. There was an over estimation of Idelvion across all concentration relative to labeled potency.

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

## B Comparison Studies:

### 1. Method Comparison with Predicate Device:

The method comparison study was conducted using the CRYOcheck Chromogenic FIX on the Instrumentation Laboratory (IL) ACL TOP 700 and IL ACL TOP 750 CTS by testing n=386 clinical samples (citrated plasma) collected from the intended use population. Results from the CRYOcheck Chromogenic FIX were compared to results from the ROX Factor IX validated laboratory developed test. The following table summarizes the line equation from the Passing-Bablok regression analysis performed for the combined dataset.

| N | FIX Activity Range (%) | Slope (95% CI) | Intercept (95% CI) | Pearson Correlation Coefficient |
| --- | --- | --- | --- | --- |
| 368 | 0.7–190.7 | 1.10 (1.08, 1.12) | 0.64 (0.20, 1.34) | 0.992 |

### 2. Matrix Comparison:

Not Applicable

## C Clinical Studies:

### 1. Clinical Sensitivity:

Not Applicable

K214002 - Page 9 of 10

{9}

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

D Clinical Cut-Off:
Not Applicable

E Expected Values/Reference Range:
The reference interval study was conducted at one laboratory site. The FIX activity of plasma samples collected from 128 normal, ostensibly healthy individuals (≥ 18 years) was tested by two operators using three lots of CRYOcheck Chromogenic FIX on two Instrumentation Laboratory ACL TOP instruments. The reference interval was established by calculating the non-parametric 95% confidence interval (2.5th to 97.5th percentiles). The calculated normal reference range for CRYOcheck Chromogenic FIX is 78.8–154.9%.

VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

K214002 - Page 10 of 10