The Wako AFP-L3% assay is intended as a risk assessment test for the development of hepatocellular carcinoma (HCC) in patients with chronic liver diseases (CLD). Elevated AFPL3% values (≥ 10%) have been shown to be associated with a seven-fold increase in the risk of developing HCC within the next 21 months. Patients with elevated serum AFPL3% should be more intensely evaluated for evidence of HCC according to the existing HCC practice guidelines in oncology.

Device Description

The Wako AFP-L3% device consists of reagent 1 (LCA and anion 1-conjugated anti-AFP mouse monoclonal antibody), reagent 2 (horseradish peroxidase (POD)labeled anti-AFP mouse monoclonal antibody and anion 2 conjugated anti-AFP mouse monoclonal antibody, substrate 1 (4 acetamidophenol in 2-propanol) and substrate 2 (hydrogen peroxide) and a column. Reagent 1, reagent 2 and the column are ready-to-use. Elution buffers A to C, sample cups, inside and outside cuvettes are sold separately from kit.

The Wako AFP-L3 Calibrator set and Control set are sold separately. The calibrator set consisted of Calibrator 1 and 2. Calibrator 1 contains human AFP -L1 fraction and Calibrator 2 has human AFP-L3 fraction. The control set consisted of Control 1 and 2, each containing different concentrations of human AFP-L1 and L-3.

AI/ML Overview

Here's an analysis of the provided 510(k) summary, extracting the requested information about acceptance criteria and the supporting study:

Acceptance Criteria and Device Performance for Wako LBA AFP-L3

1. Table of Acceptance Criteria and Reported Device Performance

The 510(k) summary provided does not explicitly define acceptance criteria in a structured table for each performance characteristic. However, it presents measured performance values and, for linearity, implicitly sets criteria within the reported ranges for slope and R². For clinical performance, the intended use statement sets a clear threshold for risk assessment.

Here's an inferred table based on the provided data:

Performance Characteristic	Acceptance Criteria (Inferred/Stated)	Reported Device Performance
Analytical Performance
Within-run Precision (%CV)	Low (Control 1): AFP <1.5%, AFP-L3 <3.6%, AFP-L3% <3.3%	AFP: 1.5%, AFP-L3: 3.6%, AFP-L3%: 3.3%
	Medium (Control 2): AFP <1.5%, AFP-L3 <3.7%, AFP-L3% <3.0%	AFP: 1.5%, AFP-L3: 3.7%, AFP-L3%: 3.0%
	High (Control 3): AFP <1.4%, AFP-L3 <1.6%, AFP-L3% <0.7%	AFP: 1.4%, AFP-L3: 1.6%, AFP-L3%: 0.7%
Total Precision (%CV)	Low (Sample 1): AFP <5.7%, AFP-L3 <9.6%, AFP-L3% <6.4%	AFP: 5.7%, AFP-L3: 9.6%, AFP-L3%: 6.4%
	Medium (Sample 2): AFP <3.7%, AFP-L3 <8.0%, AFP-L3% <6.7%	AFP: 3.7%, AFP-L3: 8.0%, AFP-L3%: 6.7%
	High (Sample 3): AFP <3.9%, AFP-L3 <5.9%, AFP-L3% <2.7%	AFP: 3.9%, AFP-L3: 5.9%, AFP-L3%: 2.7%
Reproducibility (%CV)	AFP <3.9%, AFP-L3 <6.9%	AFP: 1.0-3.9%, AFP-L3: 1.5-6.9%
Recovery	AFP: 95.1-108.4%, AFP-L3: 95.3-118.2%	AFP: 95.1-108.4%, AFP-L3: 95.3-118.2%
Linearity (Total AFP)	R² >0.99 for all sites/runs	R² ranges from 0.9974 to 0.9997
Linearity (AFP-L3%)	R² >0.98 for all sites/runs	R² ranges from 0.9835 to 0.9999
Analytical Sensitivity (MDC)	<0.8 ng/mL (claimed)	0.26 ng/mL
Functional Sensitivity	<10 ng/mL	10 ng/mL
HAMA interference (% Rec.)	AFP: 94.5-100.1%, AFP-L3: 95-112.8%, AFP-L3%: 97.9-113.5%	AFP: 94.5-100.1%, AFP-L3: 95-112.8%, AFP-L3%: 97.9-113.5%
Clinical Performance
Risk Assessment	Elevated AFP-L3% (≥ 10%) associated with a seven-fold increase in HCC risk within 21 months	Relative Risk = 7.0 (95% CI: 4.1 to 11.9) for AFP-L3% ≥10% vs. <10% (excluding Group 3)
Clinical Cut-off	AFP-L3% >10% considered positive for HCC	Confirmed as threshold for risk assessment

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

The primary clinical study involved 494 evaluable patients from:

6 US clinical sites (Lahey (MA), MCV (VA), Miami (FL), Mt. Sinai (NY), UCSF (SF), UPenn (PA))
1 Canadian clinical site (Toronto (Canada))

The study was a double-blind, multi-site prospective study. Serum samples were collected and stored frozen, and AFP-L3% tests were performed retrospectively by Wako Chemicals USA, Inc.

For the relative risk calculation, 57 patients from Group 4 (No HCC) were excluded due to less than 21 months of follow-up, resulting in a dataset of 312 patients (39 HCC, 273 No HCC) being used for the primary relative risk calculation.

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

The summary does not explicitly state the number or specific qualifications of experts used to establish the ground truth for the clinical study. However, the diagnosis of HCC for all enrolled patients was made by "at least one or a combination of the following observations," indicating clinical judgment by physician investigators:

HCC result on a liver biopsy
Enlarging mass by imaging (ultrasound, CT, MRI) with elevated serum total AFP
Enlarging mass by CT or MRI in the setting of cirrhosis
Very high serum total AFP (>400-500 ng/mL) alone
At least 3 serial blood draws showing rising serum AFP in the setting of a liver mass
Mass on CT scan enhancing in arterial phase and hypoattenuating in venous phase.

For evaluable subjects without HCC at enrollment, they were categorized by "the physician investigators" based on biopsy, explanted liver histology, and imaging results. This implies that the ground truth was established by the clinical team at each site.

4. Adjudication Method for the Test Set

The adjudication method for the test set is not explicitly described as a formal numerical system (e.g., 2+1, 3+1). Instead, the diagnosis of HCC was based on a combination of clinical observations and confirmatory tests, as determined by the "physician investigators." This suggests a consensus-based or standard clinical practice approach at each site, rather than a centralized, predefined adjudication panel for the study.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was done. This device is a quantitative immunoassay for a biomarker (AFP-L3%) and is not an imaging device or AI-assisted diagnostic tool that would typically involve human readers. The study focuses on the standalone performance of the test in assessing HCC risk.

6. Standalone Performance (Algorithm Only Without Human-in-the-Loop)

Yes, a standalone performance study was done. The entire clinical study described is a standalone evaluation of the Wako AFP-L3% assay. The device measures AFP-L3% in serum, and its performance is evaluated based on its correlation with subsequent HCC development (risk assessment). Human intervention in the diagnostic process occurred after the test result was obtained, not as part of the test interpretation itself. The AFP-L3% values and the 10% cutoff are directly used to stratify patient risk.

7. Type of Ground Truth Used

The ground truth for the clinical study was primarily established through clinical diagnosis based on a combination of pathology (liver biopsy, explanted liver histology), imaging results (CT, MRI, ultrasound), and serial biomarker measurements (total AFP), as determined by physician investigators and standard oncology practice guidelines. This represents a robust clinical ground truth.

8. Sample Size for the Training Set

The 510(k) summary does not specify a separate training set for the clinical performance evaluation. The clinical study described appears to be the primary validation study (test set) for the device's intended use claim. For the analytical performance (precision, linearity, etc.), specific samples (control sera, spiked samples, diluted samples) were used, but these are not referred to as a "training set" in the context of machine learning model development. Given this is an immunoassay, the "training" aspect is more about assay optimization and standard curve generation during assay development than a machine learning training phase.

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

As no dedicated "training set" is identified in the context of a machine learning algorithm, this question is not directly applicable. For the analytical studies, the ground truth was inherently established by:

Known concentrations in spiked samples.
Reference materials (e.g., 1st International standard for AFP from WHO) for calibrator and control value assignments.
Manufacturer-defined preparations for control sera (e.g., purified AFP-L1 and L-3 spiked into pooled serum).

Summary

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:

K041847

B. Purpose for Submission: New device
C. Analyte: AFP-L3%
D. Type of Test:

Quantitative, fluorescence enzyme immunoassay

E. Applicant: Wako Chemicals USA, Inc.

F. Proprietary and Established Names:

Wako LBA AFP-L3 Wako AFP-L3 Calibrator Set Wako AFP-L3 Control Set Wako LiBASys

G. Regulatory Information:

1. Regulation section:
- 21 CFR§866.6030, AFP-L3% immunological test system
- 21 CFR§862.1150, Calibrator
- 21 CFR&862.1660. Ouality control material (assayed and unassayed)
1. Classification:

AFP-L3% immunological test system - Class II Calibrator - Class II

Quality control material (assayed and unassayed) - Class I

1. Product Code:
  NSF, Test, Alpha Fetoprotein L3 subfraction (AFP-L3%) for hepatocellular carcinoma risk assessment
JIT, Calibrator, Secondary
JJX, Single (specified) analyte controls (assayed and unassayed)
1. Panel:
  Immunology (82), Chemistry (75)

H. Intended Use:

1. Intended Use:
  The Wako AFP-L3% assay is intended as a risk assessment test for the development of hepatocellular carcinoma (HCC) in patients with chronic liver diseases (CLD). Elevated AFPL3% values (≥ 10%) have been shown to be associated with a seven-fold increase in the risk of developing HCC within the next 21 months. Patients with elevated serum AFPL3% should be more intensely evaluated for evidence of HCC according to the existing HCC practice guidelines in oncology.
1. Indication(s) for use: Same as intended use.
1. Special condition for use statement(s):

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For prescription use only.

1. Special instrument Requirements: Use with automated analyzer LiBASys.

I. Device Description:

J. Substantial Equivalence Information:

1. Predicate device name(s): No predicate
1. Predicate K number(s): Not applicable
1. Comparison with predicate: Not applicable

K. Standard/Guidance Document Referenced (if applicable):

CLSI Guidelines EP5-A. EP7-A and EP6-A.

L. Test Principle:

Human AFP is a 70 kD glycoprotein with a single asparagine-linked carbohydrate chain. Using Lens culinaris agglutinin (LCA) which has an affinity for the carbohydrate chain that has an additional a 1-6 fucose residue bound to Nacetylgucosamine at the reducing end. AFP can be subdivided into 3 microheterogeneity forms, L1, L2 and L3. AFP L1 and L3 are major components of AFP in serum of HCC patients. AFP-L3% is the ratio of AFP with a 1-6 fucose residue to total AFP. The concentrations of LCA-reactive and LCAnonreactive AFP are measured separately using three anti-AFP monoclonal antibodies that detect different epitope, LCA and a fluorophotometric substrate. When a patient serum sample is mixed with LCA and anion 1 (sulfated tyrosine pentamer)-conjugated anti-AFP monoclonal antibody. LCA reacts with the sugar chain of LCA-reactive AFP while anion 1 conjugated anti-AFP monoclonal antibody binds to all AFP molecules forming immune complexes. When reagent 2 is added, immune complexes react with horseradish peroxidase (POD) labeled anti-AFP monoclonal antibody and anion 2 (sulfated tyrosine pentamer)conjugated anti-AFP monoclonal antibody. Anion 2- conjugated anti-AFP monoclonal antibody recognizes the base area of the sugar chain of APP and competes with previously reacted LCA on LCA-reactive AFP but binds to LCA non-reactive AFP without competitive reaction. The POD labeled anti-AFP

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monoclonal antibody binds to all the AFP molecules in the sample. Therefore, two types of immune complexes with different anionic charges are formed. Complex 1 has 5 sulfate residues and complex 2 with 13 sulfate residues. The reaction mixture is introduced to an anion-exchange column and by means of stepwise gradient; immune complex fractions are separately eluted into reaction cups. The POD activity of each complex is measured and is determined as the increase of fluorescence intensity of 5,5 '-diacetoamide-2,2'-bisphenol formed by the reaction of hydrogen peroxide and 4-acetamidophenol. The total fluorescence intensity of complex 1 and 2 represents AFP concentration and AFP-L3% is complex 1/complex 1 + complex 2. AFP concentration and AFP-L3% are obtained by comparing to standards with known AFP concentration and AFP-L3%. (see illustration below)

Step 1: Addition of LCA and anion 1-conjugated anti-AFP monoclonal antibody

[Anion 1 conjugated anti-AFP monoclonal antibody}

[LCA-reactive AFP]—[LCA }

[Anion 1 conjugated anti-AFP monoclonal antibody }

[LCA non-reactive AFP]

Step 2: Addition POD labeled anti-AFP monoclonal antibody and anion 2conjugated anti-AFP monoclonal antibody

Complex 1

[Anion 1 conjugated anti-AFP monoclonal]

[LCA-reactive AFP]—[LCA]

[POD labeled anti-AFP monoclonal]

Complex 2

[Anion 1 conjugated anti-AFP monoclonal]

[LCA non-reactive AFP]-[Anion 2-conjugated anti-AFP monoclonal]

[POD labeled anti-AFP monoclonal]

Step 3: Column separation

Step 4: Elution of immune complexes

Step 5: Addition of substrates

Step 6: Fluorescence detection

M. Performance Characteristics (if/when applicable):

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1. Analytical performance:

a. Precision/Reproducibility:
Within-run precision - Three control sera with low, medium and high concentration of total AFP were assayed 21 times. Control serum #1 was prepared by spiking purified AFP-L1 and L-3 into pool serum. Results are shown in the following table.

	Control 1			Control 2			Control 3
	AFP	AFP-L3	AFP-	AFP	AFP-L3	AFP-	AFP	AFP-L3	AFP-
	(ng/mL)	(ng/mL)	L3%	(ng/mL)	(ng/mL)	L3%	(ng/mL)	(ng/mL)	L3%
Mean	32.5	8.1	24.8	501.9	116.0	23.1	174.2	124.6	71.5
SD	0.48	0.29	0.82	7.31	4.30	0.70	2.39	2.01	0.53
CV	1.5	3.6	3.3	1.5	3.7	3.0	1.4	1.6	0.7

Total precision - Study was performed according to CLSI Guideline EP5-A. Three control sera were assayed in duplicates, two runs per day for 21 days. Results were summarized below.

	Sample 1			Sample 2			Sample 3
	AFP(ng/mL)	AFP-L3(ng/mL)	AFP-L3%	AFP(ng/mL)	AFP-L3(ng/mL)	AFP-L3%	AFP(ng/mL)	AFP-L3(ng/mL)	AFP-L3%
Total Mean	33.1	8.4	25.4	506.5	117.9	23.3	175.3	126	71.8
Within-run SD	0.50	0.38	1.01	8.89	7.98	1.29	2.90	2.7	0.73
Within-run CV (%)	1.5	4.5	4.0	1.8	6.8	5.5	1.7	2.1	1.0
Day-to-Day SD	1.14	0.29	0.55	12.72	3.44	0.53	2.86	2.67	0.70
Day-to-Day CV (%)	3.4	3.5	2.2	2.5	2.9	2.3	1.6	2.1	1.0
Run-to-Run SD	1.42	0.66	1.15	10.76	3.69	0.67	5.46	6.36	1.62
Run-to-Run CV (%)	4.3	7.9	4.5	2.1	3.1	2.9	3.1	5.0	2.3
Total SD	1.89	0.81	1.63	18.88	9.44	1.55	6.81	7.41	1.91
Total CV (%)	5.7	9.6	6.4	3.7	8.0	6.7	3.9	5.9	2.7

Reproducibility - Within-run precision studies were performed by 3 clinical laboratories using two samples with different concentrations of AFP and AFP-L3. The samples were assayed in 21 replicates each run for two runs. Percent CV for AFP and AFP-L3 ranged from 1.0% to 3.9% and 1.5% to 6.9% respectively.

Recovery - Five different concentrations of AFP [(48.6, 118.8, 288.4, 436.1 and 699.0 ng/mL) containing AFP-L3 concentrations of 9.9, 90.5, 42.4, 137.4 and 290.8 ng/mL and calculated AFP-L3% of 20.3, 76.2, 14.7, 31.5 and 41.6%] were spiked into aliquots of 3 serum samples with endogenous AFP concentrations of 15.4, 44.2 and 148.6 ng/mL [containing AFP-L3 concentrations of 0.4, 29.4 and 7.7 ng/mL]. Percent recovery for AFP and AFP-L3 ranged from 95.1% to 108.4% and 95.3% to 118.2% respectively.

b. Linearity/assay reportable range: The assay measuring range is 0.8 ng/mL to 1000 ng/mL

Linearity was evaluated according to CLSI Guideline EP6-A. For AFP, a pool serum sample was spiked with AFP-L1 and AFP-L3

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to achieve two samples, one with approximately 25% and the other 80% AFP-L3. These two samples were serially diluted with saline. Each of the seven dilutions was assayed in five replicates in two runs. For AFP-L3, three samples (50 ng/mL, 200 ng/mL and 900 ng/mL AFP-L1 and AFP-L3) were mixed in various proportions with three low AFP-L3 samples. Each sample was assayed in five replicates in two runs. The assay is linear up to 1000 ng/mL AFP.

Linearity studies were also performed in 3 clinical sites. Thirteen samples with same AFP-L3% but total AFP concentrations covering the assay linear range (0-1000 ng/mL) were analyzed in quadruplicates and for two runs. Eleven samples with same total AFP concentration but AFP-L3% covering the assay linear range (0-100%) were similarly assayed. Results for all three sites are summarized below.

Total AFP

	Site 1		Site 2		Site 3
	Run 1	Run 2	Run 1	Run 2	Run 1	Run 2
Slope	1.0074	1.0067	1.0031	1.0144	0.9921	0.9888
Intercept	-6.2226	-3.1165	-3.5117	-2.3976	-5.6467	-2.8759
R2	0.9988	0.9997	0.9994	0.9992	0.9974	0.9997

AFP-L3%

	Site 1		Site 2		Site 3
	Run 1	Run 2	Run 1	Run 2	Run 1	Run 2
Slope	1.0494	0.9958	0.9942	0.9933	0.9946	0.9992
Intercept	3.8505	-0.7228	0.3529	-0.3858	-1.3065	-1.1302
R2	0.9835	0.9995	0.9999	0.9996	0.998	0.9992

Traceability (controls, calibrators, or method): C.

The 1st International standard for AFP from WHO was used to assign values to the calibrators and controls.

The primary standards for calibrator 1 and 2 are prepared by diluting purified AFP-L1 and AFP-L3 respectively. Values are assigned using the WHO AFP standard. The primary standards are then used as calibrators to assign values to bulk preparations of AFP-L1 and AFP-L2 by assaying on four LiBASys instruments together with a previous lot of calibrators. If the bulk preparations meet the established acceptance criteria, the solutions are subdivided. One vial is selected from each of the new lot of calibrators for product testing with a previous lot of calibrators and using the primary standards for calibration. AFP values of the new lot are calculated from the mean results of both bulk and product tests. The AFP-L3% of calibrator 1 is assigned 0% and that of calibrator 2 is assigned as 100%.

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The two AFP-L3 controls are prepared by mixing purified AFP-L1 and AFP-L3 and assayed on 5 LiBASys instruments using the primary standards 1 and 2 for calibration and compared to previous lot of AFP-L3 control set. Procedures for value assignment and product testing are similar to that described for calibrators.

d. Detection limit (functional sensitivity):

The minimal detectable concentration (MDC) for AFP was determined by assaying 21 replicates of a deionized water sample and is defined as mean concentration of AFP +2SD. Data showed the mean AFP concentration was 0.14 ng/mL with a SD of 0.06 ng/mL. The MDC was therefore 0.26 ng/mL. The analytical sensitivity claim is 0.8 ng/mL.

Functional sensitivity was determined by assaying 11 serum samples with AFP-L1 and AFP-L3 concentrations between 0 and 65 ng/mL. For each concentration, 8 replicates were run and the percents CV obtained of the replicates were plotted against the respective AFP-L1 or L3 concentrations. Functional sensitivity was established to be 10 ng/mL.

e. Analytical specificity:

Interference was determined by spiking different concentrations of each interfering substance into aliquots of a low and a high AFP serum sample. The AFP samples were prepared by spiking in AFP-L1 and L-3 into a pooled serum. The interfering substances tested include hemoglobin (0-1000 mg/dL), free bilirubin (0-40 mg/dL), conjugated bilirubin (0-30 mg/dL), intrafat (0-2%), rheumatoid factor (0-550 IU/mL), glucose (0-1000 mg/dL), galactose (0-200 mg/dL) and ascorbate (0-50 mg/dL). Glucose concentration >600 mg/dL can result in decreased AFP-L3%. Decreased AFP-L3% results were also observed for free and conjugated bilirubins >20 mg/dL (this information is in the package insert).

Drug interference was determined by additive studies of vitamins B1, B6 and B12, interferons (IFN) a, B and y, ibuprofen, acetaminophen and acetylsalicylic acid. Results of AFP and AFP-L3% were not affected by Vitamins B1 (14 mg/dL), B6 (25 mg/dL), B12 (50 mg/dL), IFN-α (3,000 units/mL), IFN-β (3,000 units/mL), IFN-y (3,000 JRU/mL), Ibuprofen (40 mg/dL), acetaminophen (20 mg/dL) and acetylsalicylic acid (50 mg/dL). Percent recoveries ranged from 95.2% to 105.6%.

HAMA interference - Four different concentrations of AFP [(19.4 ng/mL, 71.1 ng/mL, 238.1 ng/mL and 297.5 ng/mL) containing

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AFP-L3 concentrations of 3.7, 58.3, 84.5 and 51.5 ng/mL] were spiked into aliquots of a positive HAMA serum sample. Each AFP concentration was assayed in quadruplicate and percent recoveries were determined for AFP, AFP-L3 and AFP-L3%. Percent recoveries ranged from 94.5% to 100.1% for AFP, 95% to 112.8% for AFP-L3 and 97.9% to 113.5% for AFP-L3%.

f. Assay cut-off:
AFP-L3% is not reported if total AFP is <10 ng/mL. Data supporting the cut-off were from published literature (Oka, H., et al. J. Gastroenterol. Hepatol. 16 (2001), 1378-1383 and Yamagata, Y., et al. Clin. Chim. Aceta 327 (2003), 59-67).
1. Comparison studies:
- a. Method comparison with predicate device: No predicate device.
- b. Matrix comparison:

Not applicable since only serum is the only matrix.

1. Clinical studies:
- Clinical sensitivity: a. Not applicable.
- Clinical specificity: b. Not applicable.
- Other clinical supportive data (when a and b are not applicable) C. Longitudinal data analysis

This is a double-blind, multi-site prospective study. Four hundred ninety six patients with liver disease were enrolled at 7 clinical sites (6 US and one Canadian). Data were collected for serum chemistries and imaging (CT, MRI, and/or ultrasound), presenting symptoms, medications, interventions, therapeutics, and other medical information required for patient management. Serum samples were collected and stored frozen. All AFP-L3% tests were performed by Wako Chemicals USA. Inc.

At the time of enrollment, subjects belonged to two categories newly diagnosed HCC (a minimum of 50 HCC patients with tumor size <5 cm for informational purposes) or did not have HCC. The diagnosis of HCC for all patients enrolled was made by at least one or a combination of the following observations: 1) a HCC result on a liver biopsy, 2) an enlarging mass, typically greater than 2 cm by imaging (includes ultrasound, CT and MRI) with elevated serum total AFP (typically 20-200 ng/mL), 3) an enlarging mass by CT or MRI in the setting of cirrhosis. 4) a very high serum total AFP >400-500 ng/mL alone, 5) at least 3 serial blood draws that showed a rising serum AFP in the setting of a liver mass or 6) a mass on CT

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scan that enhances in the arterial phase and was hypoattentuating compared to the rest of the liver in the venous phase.

The objective of the study was to observe a minimum of 30 subjects who developed verifiable HCC during the study. At the end of the study, all evaluable subjects without HCC at enrollment were categorized by the physician investigators based on biopsy, explanted liver histology, imaging results and site policies into three groups: 1) developed confirmed HCC during study and with lesions of at least 0.5 cm in diameter; 2) suspected of possibly HCC with lesions at least 0.3 cm in diameter and high total AFP results and 3) did not have HCC but had complete workup. Data were collected on study subjects for the duration of the study (approximately 3 years) or until death, developed HCC or end of study. Evaluable patients were defined as patients who had at least one valid AFP-L3% result.

In the data analysis, the number of days was calculated for each patient as the time between the first AFP-L3% ≥ 10 and the confirmed HCC diagnosis for group 2 patients and for the other groups, the AFP-L3 % value between study enrollment and end of study was used. The primary endpoint is the relative risk (with 95% CI) of developing HCC within the next 21 months in patients with AFP-L3% ≥10% as compared to the risk in patients with AFPL3% <10%.

The number of patients screened was 582 with 76 failed screening, 10 with no samples, 53 died during study period of other causes. Two subjects in Group 4 were less than 40 years old and were excluded from the analysis. The following table summarizes the distribution of the 494 subjects by group and clinical site. One site recruited 50% of patients. Eighty-one subjects had only one sample drawn and 415 patients had more than one sample.

	Group 1	Group 2	Group 3	Group 4
	HCC	HCC Dxduring study	Suspicious	No HCC
Clinical study site					Total
Lahey (MA)	1	3	3	43	50 (10.1%)
MCV (VA)	0	3	2	53	58 (11.7%)
Miami (FL)	7	3	2	46	58 (11.7%)
Mt. Sinai (NY)	43	27	62	118	250 (50.6%)
Toronto (Canada)	1	2	1	39	43 (8.7%)
UCSF (SF)	2	1	1	14	18 (3.6%)
UPenn (PA)	0	0	0	17	17 (3.4%)
Total	54	39	71	332	494

The following table summarizes sample collection interval by number of days for patients from Group 2, 3 and 4.

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		Group 2	Group 3	Group 4
Total Number of Patients		39	71	332
Number of Samples	1	1	6	63
	2	4	7	56
	3	9	13	51
	4	5	16	48
	≥5	20	29	114
Average Number of Samples		4.5	4.3	3.6
Day Interval	Average	134.6	136.7	143.0
	Range	63.7-335.5	60.5-467	53.0-579

Demographics

The subjects consisted of 368 males and 128 females. The gender distribution between group 2 and group 4 was similar (males: group 2 = 79.5% and group 4 = 72.3%; females: group 2 = 20.5% and group 4 = 27.7%).

		Group 1	Group 2	Group 3	Group 4
		HCC	HCC Dx during study	Suspicious	No HCC	Total
Male	N	46	31	51	240	366
	%	85.2	79.5	71.8	72.3	74.1
Female	N	8	8	20	92	128
	%	14.8	20.5	28.2	27.7	25.8
Total	N	54	39	71	332	494

The overall mean age was 52.6 y (SD = 7.0 y) and with similar distribution between group 2 (mean age = 53 y, SD = 6 y) and group 4 (mean age = 52.1 y, SD = 7.1 y).

		Group 1	Group 2	Group 3	Group 4
			HCC Dxduring study
		HCC		Suspicious	No HCC	Total
Male	Mean Age	55.1	52.6	51.8	51.1	51.8
	SD	7.6	5.4	6.1	6.1	6.3
	Range	40-70	42-70	42-69	36-70	40-70
Female	Mean Age	57.3	54.5	53.2	55.2	55.0
	SD	6.3	8.0	6.2	8.4	7.9
	Range	48-66	46-66	43-67	40-70	40-70
Total	Mean Age	55.4	53.0	52.2	52.1	52.6
	SD	7.4	6.0	6.1	7.1	7.0
	Range	40-70	42-70	42-69	36-70	36-70
	N	54	39	71	332	494

The following table summarizes the ethnic distribution of the patient groups:

		Group 1	Group 2	Group 3	Group 4
		HCC	HCC Dxduring study	Suspicious	No HCC	Total
Caucasian	N	29	29	37	231	326
Caucasian	%	53.7	74.4	52.1	70.0	66.0

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		Group 1	Group 2HCC Dxduring study	Group 3	Group 4
		HCC		Suspicious	No HCC	Total
Asian	N	2	2	2	21	27
	%	3.7	5.1	2.8	6.4	5.5
Black	N	7	4	14	33	58
	%	13.0	10.3	19.7	10.0	11.7
Hispanic	N	142	2	12	39	67
	%	25.9	5.1	16.9	11.8	13.6
Other	N	2	2	6	6	16
	%	3.7	5.1	8.5	1.8	3.2
Total	N	54	39	71	330	494

The following table summarizes the history of alcoholism of the patient groups:

		Group 1	Group 2HCC Dxduring study	Group 3	Group 4
		HCC		Suspicious	No HCC	Total
Heavy	N	4	1	7	20	326
	%	9.1	2.7	10.6	6.3	6.9
Moderate	N	9	14	12	35	70
	%	20.5	16.2	28.8	17.6	19.8
Occasional	N	11	6	19	56	92
	%	25.0	16.2	28.8	17.6	19.8
No	N	20	16	28	207	271
	%	45.5	43.2	42.4	65.1	58.3
No data	N	10	2	5	14	29
Have data	N	44	37	66	318	465
Total	N	54	39	71	332	494

Similar distribution was observed between group 2 and group 4 for HBV and HVC status. Forty-six patients were HBV positive with 10.3% in group 2 and 9.0% in group 4. Of the 331 HCV positive patients, 71.8% in group 2 and 70.5% group 4. For the 119 HBV and HCV positive patients, 17.9% in group 2 vs. 20.5% in group 4.

		Group 1	Group 2	Group 3	Group 4
			HCC Dxduring study
		HCC		Suspicious	No HCC	Total
Only HBV	N	10	4	2	29	45
positive	%	18.5	10.3	2.8	8.8	9.1
Only HCV	N	26	28	43	233	330
positive	%	48.1	71.8	60.6	70.6	66.8
HBV and	N	18	7	26	68	119
HCV positive	%	33.3	17.9	36.6	20.5	24.0
HBV and	N	0	0	0	0	0
HCV negative	%	0	0	0	0	0
Total	N	54	39	71	332	494

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Disease stage according to Child's Classification was available for 85.6% (424/496) of patients with cirrhosis. Group 2 had more Grade C whereas group 4 had more Grade A. The number of patients with Grade B was similar (see below)

		Group 1	Group 2	Group 3	Group 4
			HCC Dx
		HCC	during study	Suspicious	No HCC	Total
Grade A	N	11	8	12	118	149
	%	28.9	20.5	17.6	42.6	65.4
Grade B	N	18	19	43	113	193
	%	47.4	48.7	63.2	40.8	45.7
Grade C	N	9	12	13	46	80
	%	23.7	30.8	19.1	16.6	19.0
Total	N	38	39	71	277	422

Comparison of AFP-L3% between the 4 cohorts by Mann-Whitney Test is shown below.

	Group 1(Max AFP-L3%beforetreatment)	Group 2(AFP-L3% atstart of study)	Group 2(Max AFP-L3%beforetreatment)	Group 3(within ± 3months ofenrollment)	Group 4(within ± 3months ofenrollment)
Total #patients	54	39	39	71	332
AverageAFP-L3%	11.1	10.19	17.3	6.0	3.1
AFP-L3%SD	17.7	15.99	19.0	12.8	11.2
P value vs.Group 4	<0.0001	<0.0001	<0.0001	<0.0001

Relative risk:

Relative risk was calculated using group 2 and 4. Due to the change in the intended use claim from 12 months to 21 months, 57 patients from Group 4 who had less than 21 months follow-up (total or after a positive AFP-L3% ≥ 10) were excluded from the calculation. The following table summarizes the distribution of patients with AFP-L3% ≥10% and those with AFP-L3% <10% in these groups.

	HCC	No HCC	Total
AFP-L3% ≥10%	20	21	41
AFP-L3% <10%	19	252	271
Total	39	273	312

Risk of HCC for AFP-L3% ≥10% = 48.8% (95% CI: 33.5%-64.1%) Risk of HCC for AFP-L3% <10% = 7.0% (95%CI: 4.0%-10.1%) Relative Risk = 7.0 (48.8/7.0) (95% CI: 4.1 to 11.9)

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Based on information available to the physicians, definitive diagnosis could not be made for patients in Group 3 at the time of the study and therefore were not included in the above risk calculation. Since this group accounted for 16% of the data, one has to consider the possibility of spectrum bias by excluding Group 3 in the risk analysis. The following tables showed the worst case and best possible case scenarios to determine the effect of this group in the risk estimation,

(a) Worst case scenario

	HCC	No HCC	Total
AFP-L3% ≥10%	20	$21+12 = 33$	53
AFP-L3% <10%	$19+59 = 78$	252	330
Total	98	285	383

Risk of HCC for AFP-L3% ≥10% = 37.7% (20/53) Risk of HCC for AFP-L3% <10% = 23.6% (78/330) Relative Risk = 1.6 (37.7/23.6) (95% CI: 1.1 to 2.4)

(b) Best case scenario

	HCC	No HCC	Total
AFP-L3% ≥10%	$20+12 = 32$	21	53
AFP-L3% <10%	19	$252+59 = 311$	330
Total	51	332	383

Risk of HCC for AFP-L3% ≥10% = 60.4% (32/53) Risk of HCC for AFP-L3% <10% = 5.8% (19/330) Relative Risk = 10.5 (60.4/5.8) (95% CI: 6.4 to 17.1)

Relative risks for patients positive for HBV or HCV infection were also analyzed. The following table shows the relative risk calculated for HBV positive patients in Group 2 was compared to those in Group 4.

	HCC	No HCC	Total
AFP-L3% ≥10%	1	2	3
AFP-L3% <10%	3	28	31
Total	4	30	34

Risk of HCC for AFP-L3% ≥10% = 33.3% Risk of HCC for AFP-L3% <10% = 9.7% Relative Risk = 3.4 (95% CI: 0.5 to 23.7)

Relative risk for HCV positive patients in Group 2 compared group 4 (see below).

{12}------------------------------------------------

	HCC	No HCC	Total
AFP-L3% ≥10%	14	13	27
AFP-L3% <10%	14	221	235
Total	28	234	262

Risk of HCC for AFP-L3% ≥10% = 51.9% Risk of HCC for AFP-L3% <10% = 6.0% Relative Risk = 8.7 (95% CI: 4.7 to 16.3)

Logistic regression models were run to determine the interaction effects of hepatitis virus group (HBV, HCV and both HBV and HCV), number of days and AFP-L3% values on development of HCC. The odds ratios between AFP-L3% and development of HCC were not statistically different (p=0.52) among the hepatitis virus groups. Excluding the interaction term between AFP-L3% and hepatitis virus group, the odds ratio between AFP-L3% and development of HCC, adjusted for days and hepatitis virus group was 21.8 (95% CI 8.9, 53.4) which is greater than the odds ratio adjusted for days alone (20.9, 95% CI 8.6, 50.3).

Clinical cut-off: 4.
AFP-L3% >10% considered positive for HCC.
1. Expected values/Reference range:
  Expected value range for AFP in healthy adults is 0.1 to 5.8 ng/mL according to published literature (Masseyeff R., et al. N. Engl. J. Med. 291 (1974), 532). Normal reference value for AFP-L3% is not detectable.

N. Instrument Name:

Wako LiBASys

O. System Descriptions:

1. Device Description:
  The LiBASys consists of a main analyzer, a bar code reader and a drain port. The main analyzer contains the following units: analysis, reaction, solution feed, column setup, photometric, display operation, elution accommodation, tank accommodation and power.
1. Principles of Operation: See Test Principles
1. Modes of Operation: Automated fluorescence analyzer
Software: ধ FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types: Yes
રું છ Specimen Identification: Bar-coding of the serum samples is done before the samples are loaded into the instrument

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1. Specimen Sampling and Handling:
  Serum samples are to be assayed immediately after blood collection. If not, store samples at -80°C. Patient serum samples are put into sample cups which are loaded into the sample disk housed in the analysis unit with the reaction disk.
1. Assay Types:
  Liquid binding assay using anion column separation of immune complexes.
1. Reaction Types: Rate assay.
1. Calibration:

A two-point linear calibration is required every assay run.

1. Quality Control: Two level controls are used. Values should be within ±15% of assigned values.
P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above: None.
Q. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

R. Conclusion:
The petition for Evaluation of Automatic Class III Designation for this device is accepted. The device is classified as Class II under regulation 21 CFR 866.6030 with special controls. The special control guidance document "AFP-L3% Immunological Test System" is available at WWW.fda.gov/cdrh/oivd/guidance/1570.pdf

Regulation Number and Section

§ 866.6030 AFP-L3% immunological test system.

(a)
Identification. An AFP-L3% immunological test system is an in vitro device that consists of reagents and an automated instrument used to quantitatively measure, by immunochemical techniques, AFP and AFP-L3 subfraction in human serum. The device is intended for in vitro diagnostic use as an aid in the risk assessment of patients with chronic liver disease for development of hepatocellular carcinoma, in conjunction with other laboratory findings, imaging studies, and clinical assessment.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: AFP-L3% Immunological Test Systems.” See § 866.1(e) for the availability of this guidance document.