Cryocheck Chromogenic Factor VIII

K193204 · Precision Biologic · GGP · Jul 17, 2020 · Hematology

Device Facts

• Record ID: K193204
• Device Name: Cryocheck Chromogenic Factor VIII
• Applicant: Precision Biologic
• Product Code: GGP · Hematology
• Decision Date: Jul 17, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7290
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

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

Device Story

Quantitative chromogenic assay for Factor VIII (FVIII) activity in 3.2% citrated human plasma; used in clinical laboratories. Input: patient plasma sample. Principle: two-stage reaction; stage 1 activates FVIII to FVIIIa, which works with FIXa to activate FX; stage 2 adds FXa-specific chromogenic substrate; FXa hydrolyzes substrate to release p-Nitroaniline (pNA). Output: spectrophotometric absorbance at 405 nm proportional to FVIII activity; results reported as percent activity (100% = 1.0 IU/mL). Operation: performed on Instrumentation Laboratory (IL) ACL TOP CTS and ACL TOP 50 CTS series analyzers. Healthcare providers use results to identify FVIII deficiency and manage hemophilia A patients.

Clinical Evidence

Bench testing only. Precision/reproducibility evaluated via single-site (n=80/sample) and multisite (n=90/sample) studies. Linearity established from 0–200% FVIII activity. Analytical specificity confirmed against common endogenous/exogenous substances (hemoglobin, lipids, bilirubin, heparin, etc.). Limit of Quantitation (LoQ) determined to be 0.5% FVIII activity. Method comparison against predicate (n=318) showed high correlation (r=0.994). Reference range established using 120 healthy individuals (43.2–159.3%).

Technological Characteristics

Quantitative chromogenic assay. Reagents: Bovine FX, fibrin polymerization inhibitor, human FIIa, human FIXa, calcium chloride, phospholipids, FXa substrate, Tris buffer, BSA, heparin antagonist. Sensing: Spectrophotometric absorbance at 405 nm. Connectivity: Integrated with IL ACL TOP CTS/50 CTS series analyzers. Storage: ≤-70°C. Software: Embedded instrument control software for automated analysis.

Indications for Use

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

Predicate Devices

• Coatest Sp FVIII (K042576)

Reference Devices

• Coatest Factor VIII (K833892)
• IL ACL TOP CTS analyzer (K160276)
• IL ACL TOP 750 (K150877)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K193204

B Applicant

Precision BioLogic

C Proprietary and Established Names

CRYOcheck Chromogenic Factor VIII

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 VIII activity (%)

C Type of Test:

Quantitative chromogenic assay

## III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

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CRYOcheck Chromogenic Factor VIII is for clinical laboratory use in the quantitative determination of factor VIII activity in 3.2% citrated human plasma. It is intended to be used in identifying factor VIII deficiency and as an aid in the management of hemophilia A 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:

Instrumentation Laboratory (IL) ACL TOP CTS Series and ACL TOP 50 CTS Series

## IV Device/System Characteristics:

### A Device Description:

CRYOcheck Chromogenic Factor VIII is used for determination of FVIII activity and contains the following four components:

| Reagent Name | Format (per vial set; each box contains 4 vials sets) | Description |
| --- | --- | --- |
| Reagent 1 | 1 x 1.25 mL Frozen | Bovine FX and a fibrin polymerization inhibitor, with activators and stabilizers. |
| Reagent 2 | 1 x 1.25 mL Frozen | Human FIIa, human FIXa, calcium chloride and phospholipids. |
| Reagent 3 | 1 x 1.25 mL Frozen | FXa substrate containing EDTA and a thrombin inhibitor. |
| Diluent Buffer | 1 x 7.0 mL Frozen | Tris buffer solution containing BSA and a heparin antagonist. |

### B Principle of Operation:

The CRYOcheck Chromogenic FVIII is a two-stage factor VIII (FVIII) assay. In the first stage, patient plasma (containing an unknown amount of functional FVIII) is diluted with Diluent Buffer and combined with Reagents 1 and 2, containing Factors IXa and X, thrombin, calcium, and phospholipids. The FVIII in the patient's plasma is activated and works in concert with FIXa to cause the activation of FX to FXa. Following an incubation period to allow activation of FX to FXa, the second stage of the assay occurs through the addition of the chromogenic FXa Substrate to this mixture. The FXa, present from the previous step, hydrolyzes the substrate into a peptide and p-Nitroaniline (pNA). The color produced by the release of pNA is measured spectrophotometrically at 405 nm and is proportional to the Factor VIII in the sample. FVIII results are reported in percent activity where 100% FVIII activity is equivalent to 1.0 IU/mL.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

Coatest Sp FVIII

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B Predicate 510(k) Number(s):

K042576

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K193204 | K042576 |
| --- | --- | --- |
| Device Trade Name | CRYOcheck Chromogenic FVIII | Coatest SP FVIII |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | CRYOcheck Chromogenic Factor VIII is for clinical laboratory use in the quantitative determination of factor VIII activity in 3.2% citrated human plasma. It is intended to be used in identifying factor VIII deficiency and as an aid in the management of hemophilia A in individuals aged 2 years and older. For in vitro diagnostic use. | Coatest SP FVIII is intended for photometric determination of factor VIII activity in citrated plasma, such as when identifying factor VIII deficiency or monitoring patients on replacement therapy, as well as for potency estimation of FVIII concentrates. For in vitro diagnostic use. |
| Classification | Class II | Class II |
| Type of Test | Quantitative | Quantitative |
| Measurand | Human Factor VIII activity | Human Factor VIII activity |
| Device Description | CRYOcheck Chromogenic Factor VIII is used for determination of FVIII activity and contains the following four components, packaged in glass vials and provided frozen to preserve the integrity of the components:
Reagent 1: Bovine FX and a fibrin polymerization inhibitor, with activators and stabilizers.
Reagent 2: Human FIIa, human FIXa, calcium chloride and phospholipids.
Reagent 3: FXa substrate containing EDTA and a thrombin inhibitor. | Coatest SP FVIII is a modified version of Coatest Factor VIII (K833892) reformulated to European Pharmacopoeia Standards. Coatest SP FVIII is a photometric assay containing a chromogenic substrate, S-2765, with EDTA added as a preservative, lyophilized bovine factors IXa and X with bovine albumin added as a stabilizing agent. The device also contains calcium chloride, Tris buffer stock solution containing sodium chloride, bovine serum albumin with added |

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| | Diluent Buffer: Tris buffer solution containing 1% BSA and a heparin antagonist. | antimicrobial in addition to a mixture of highly purified synthetic phospholipids. |
| --- | --- | --- |
| Test Principle | In the presence of calcium and phospholipids, FX is activated to factor Xa by FIXa. This generation is greatly stimulated by FVIII, which may be considered as a cofactor in this reaction. By using optimal amounts of Ca^{2+} and phospholipids and an excess of FIXa and FX, the rate of activation of FX is solely dependent on the amount of FVIII. FXa hydrolyses the chromogenic substrate S- 2765 thus liberating the chromophoric group, pNA. The color is then read photometrically at 405 nm. The generated FXa and thus the intensity of color are proportional to the FVIII activity in the sample. Hydrolysis of S-2765 by thrombin formed is prevented by the addition of the synthetic thrombin inhibitor, I- 2581, together with the substrate. | In the first stage of the chromogenic assay, test plasma (containing an unknown amount of functional FVIII) is added to a reaction mixture comprised of calcium, phospholipids, human purified thrombin and FIXa and bovine FX (Reagent 1 and Reagent 2). This mixture swiftly activates FVIII to FVIIIa, which works in concert with FIXa to activate FX. When the reaction is stopped, FXa production is assumed to be proportional to the amount of functional FVIII present in the sample. The second stage of the assay is to measure FXa through cleavage of an FXa-specific peptide nitroanilide substrate (FXa Substrate). P-nitroaniline is produced, giving a color that can be measured spectrophotometrically by absorbance at 405 nm. |
| Expression of Results | Quantitative; results are expressed as percent activity interpreted relative to a calibration curve. | Same |
| General Device Characteristic Differences | | |
| Instrument(s) | IL ACL TOP CTS Series and ACL TOP 50 CTS Series | Manual method, IL ACL 9000 |
| Calibrator and control plasmas | CRYOcheck Reference Control Normal
CRYOcheck Abnormal 1 Reference Control
CRYOcheck Abnormal 2 Reference Control
CRYOcheck Reference Control | HemosIL Normal Control (Assayed)
HemosIL High Abnormal Control (Assayed)
Calibration plasma calibrated against an International Standard |

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| | Normal, CRYOcheck Abnormal 1 Reference Control, and CRYOcheck Abnormal 2 Reference Control are traceable to the WHO International Standard for Factor VIII/VWF | |
| --- | --- | --- |
| Storage | ≤-70°C until expiration | 2–8°C until expiration |
| Assay Reportable Range | 0–200% FVIII activity | 0–150% FVIII activity |
| Reference Range | 43.2–159.3% FVIII activity | Manual Method: 48.6–126% Instrument Application: 55.4–148.9% |
| Limit of Detection | 0.5% FVIII activity | 1% FVIII activity |

VI Standards/Guidance Documents Referenced:

- CLSI EP07 3rd Edition, Interference Testing in Clinical Chemistry; Approved Guideline, Third Edition, April 2018.
- CLSI EP06, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline, April 2003.
- CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Procedures; Approved Guideline, Third Edition, October 2014.
- CLSI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline, Second Edition, June 2012.
- CLSI EP25-A, Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline, September 2009.
- CLSI EP28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline, Third Edition, October 2010.

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:

a. Single-site Study:

This study was conducted at one internal site over 20 days with two runs per day and two replicates per run, for a total of 80 determinations per sample per lot. The study design included a single representative instrument model, three reagent lots, one normal and two abnormal reference controls, as well as five patient plasma samples representing very low, low, mid, normal and high levels of FVIII activity. Precision estimates were calculated for each of the following variance components: within-run, between-run,

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between-day, between-lot and total imprecision. The results for within-run and total imprecision for all lots combined are provided in the summary table below.

| Sample | N | Mean (%) | Within-Run | | Within-Laboratory | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV |
| Normal reference control | 240 | 80.8 | 3.3 | 4.1 | 4.0 | 5.0 |
| Abnormal 1 reference control | 240 | 26.1 | 1.5 | 5.6 | 1.9 | 7.1 |
| Abnormal 2 reference control | 240 | 7.8 | 0.7 | 8.4 | 0.8 | 9.9 |
| Plasma sample 1 | 240 | 1.0 | 0.1 | 8.2 | 0.1 | N/A |
| Plasma sample 2 | 240 | 5.4 | 0.4 | 6.6 | 0.4 | 7.4 |
| Plasma sample 3 | 240 | 26.0 | 1.4 | 5.4 | 1.7 | 6.7 |
| Plasma sample 4 | 240 | 85.3 | 3.5 | 4.1 | 4.3 | 5.1 |
| Plasma sample 5 | 240 | 152.1 | 4.9 | 3.2 | 5.7 | 3.8 |

# b. Multisite Study

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

| Sample | N | Mean (%) | Within-Run | | Between-Site | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV |
| Normal reference control | 270 | 80.3 | 3.6 | 4.5 | 0.0 | 0.0 | 4.1 | 5.1 |
| Abnormal 1 reference control | 270 | 25.4 | 1.3 | 5.2 | 1.5 | 5.7 | 2.1 | 8.2 |
| Abnormal 2 reference control | 270 | 7.7 | 0.5 | 7.0 | 0.4 | 4.9 | 0.7 | 9.6 |
| Very low plasma sample | 270 | 1.1 | 0.1 | N/A | 0.0 | N/A | 0.2 | N/A |
| Normal plasma sample | 270 | 85.4 | 4.3 | 5.1 | 0.0 | 0.0 | 5.0 | 5.8 |
| High plasma sample | 270 | 156.9 | 7.8 | 5.0 | 6.1 | 3.9 | 11.4 | 7.3 |

# 2. Linearity/assay reportable range:

Linearity studies were performed following the CLSI EP06-A guideline using three reagent lots on a representative instrument model. Fifteen sample dilutions were prepared using a high FVIII (260%) and a congenital FVIII deficient plasma sample (0%). Each dilution was tested in four replicates. Based on the results of the linearity study, the claimed linear range was determined to be 0–200%.

# 3. Analytical Specificity/Interference:

Interference studies were conducted based on the CLSI EP07-A2 guideline. The study design included a single reagent lot and one representative instrument model. Potentially interfering endogenous and exogenous substances were spiked into the test samples (one normal and one

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abnormal reference control plasma sample) and the control samples. None of the substances in the following table (endogenous or exogenous substances) were found to lead to clinically significant interference.

| Interfering Substance | Concentration Tested |
| --- | --- |
| Hemoglobin | ≤ 500 mg/dL |
| Intralipid | ≤ 500 mg/dL |
| Unconjugated bilirubin | ≤ 29 mg/dL |
| vWF | ≤ 20 μg/mL |
| Unfractionated heparin | ≤ 2 IU/mL |
| Low molecular weight heparin | ≤ 2 IU/mL |
| Fondaparinux | ≤ 1.25 mg/L |
| Lupus anticoagulant | ≤ 1.8 dRVVT ratio |

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 parameters values in the borderline pathological range 30% to 40% and 5% to 10%, respectively. Calibrator value assignments are traceable to the WHO International Standard for Factor VIII/VWF (6th IS Factor VIII/VWF Plasma, 07/316).

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 (15–25°C) and below -70°C. The study was performed using one reagent lot of CRYOcheck Chromogenic Factor VIII on IL ACL TOP 300 and IL ACL TOP 700 (K160276) analyzers with four replicate measurements at each time point for each sample. The study data demonstrate that citrated plasma samples are stable for two hours when stored at room temperature (15–25°C), three months when stored at <-70°C, including up to two freeze thaw cycles.

5. Detection Limit:

The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) for the test system was determined following the CLSI EP17-A2 guideline. Each study design included three reagent lots and one representative instrument model.

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The limit of blank (LoB) was determined using four plasma samples obtained from individuals with severe congenital Hemophilia A. Each sample was tested in three replicates on five different days on one representative instrument model for n=15 determinations per reagent lot. The sponsor determined the LoB to be 0.4% FVIII activity.

The limit of detection (LoD) was determined using four plasma samples obtained from individuals with congenital Hemophilia A. Each sample was tested in three replicates on five different days on one representative instrument model for n=15 determinations per reagent lot. The sponsor determined the LoD to be 0.5% FVIII activity.

The limit of quantitation (LoQ) was determined using four plasma samples with low FVIII activity at the following target concentrations: 0.4% FVIII, 0.4% FVIII, 0.5% FVIII and 0.5% FVIII. Each of the four plasma samples were tested in three replicates per day on five different days, for n=15 determinations per reagent lot. The sponsor determined the LoQ to be 0.5% FVIII activity.

6. Assay Cut-Off:

Not applicable

B Comparison Studies:

1. Method Comparison with Predicate Device:

The method comparison study was conducted using the CRYOcheck Chromogenic Factor VIII on the Instrumentation Laboratory ACL TOP CTS and ACL TOP 750 CTS by testing n=318 clinical samples (citrated plasma) collected from the intended use population. Results from the CRYOcheck Chromogenic Factor VIII were compared to results from the Instrumentation Laboratory Coatest SP FVIII on the Instrumentation Laboratory ACL TOP 700. The following tables summarize the line equation from the Passing-Bablok regression analysis performed for the combined dataset and the absolute predicted bias.

| N | Pearson Correlation Coefficient | Slope (95% CI) | Intercept (95% CI) |
| --- | --- | --- | --- |
| 318 | 0.994 | 1.041 (1.027, 1.058) | 0.720 (0.252, 1.205) |
| %FVIII activity | Absolute Predicted Bias (95% CI) |
| --- | --- |
| 1 | -1.11 (-1.87, -0.35) |
| 5 | -0.81 (-1.53, -0.08) |
| 45 | 2.20 (1.71, 2.69) |
| 50 | 2.57 (2.09, 3.06) |

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| 100 | 6.33
(5.51, 7.15) |
| --- | --- |
| 150 | 10.09
(8.71, 11.47) |

2. **Matrix Comparison:**
Not applicable

**C Clinical Studies:**

1. **Clinical Sensitivity:**
Not applicable

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 FVIII activity of plasma samples collected from 120 normal, ostensibly healthy individuals (≥ 18 years) was tested by two operators using three lots of CRYOcheck Chromogenic Factor VIII on an Instrumentation Laboratory ACL TOP CTS. 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 Factor VIII is 43.2–159.3%.

**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.

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