This in vitro diagnostic procedure is a solid- phase enzyme immunoassay intended for the quantitative determination of CK-MB in human serum or plasma on the Technicon Immuno 1 system. When used in combination with other clinical data such as presenting symptoms and EKG values, measurement of CK-MB aids in the diagnosis of acute myocardial infarction.

Device Description

The assay is an enzyme label sandwich assay using two monoclonal antibodies. A CK-MB specific antibody is labelled with fluorescein and the Fab' fragment of an antibody specific for the B subunit is labelled with alkaline phosphatase( ALP) The solid phase consists of a suspension of magnetizable particles coated with antibody to fluorescein ( IMP reagent). Sample or calibrator. R1 reagent containing fluorescein - antibody conjugate, R2 reagent containing ALP-antibody conjugate and IMP reagent are mixed and incubated at 37°C. In the presence of CK-MB a fluorescein-conjugate: CK-MB: ALP-conjugate complex is formed and captured by the anti fluorescein antibodies on the magnetic particles. The particles are washed and pNPP (paranitrophenyl phosphate) substrate is added. The ALP in the antibody conjugate reacts with the pNPP to form para-nitrophenoxide and phosphate. Increasing absorbance due to the formation of paranitrophenoxide is monitored at 405 nm and 450 nm. The dose response curve is directly proportional to the concentration of CK-MB in the sample. A quadratic fit through zero is used to construct the dose response curve.

The assay has a range of 0 to 300 ng/ml and calibrators are provided with values of 0, 5, 10, 30, 100 and 300 ng/ml.

All the results reported herein were obtained by using a quadratic fit through zero algorithm to construct the standard curve.

AI/ML Overview

Here's an analysis of the provided text regarding the acceptance criteria and study for the Immuno 1 CK-MB method:

Understanding the Context:

This document describes the performance of the Immuno 1 CK-MB method, an in vitro diagnostic procedure for measuring CK-MB in human serum or plasma. The primary focus of the provided sections is to demonstrate the comparability and stability of results when using plasma samples as opposed to serum, and to establish storage guidelines.

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state formal "acceptance criteria" in a tabulated format with pass/fail thresholds. Instead, it presents performance data (regression analysis, imprecision, and storage stability) and implicitly suggests that the observed performance validates the use of plasma samples under specified conditions.

Based on the provided data and the intent to show plasma is comparable and stable, here's a table reflecting implied acceptance or expected performance:

Performance Metric	Implied/Observed Acceptance Criteria (for plasma as alternative to serum)	Reported Device Performance (Plasma vs. Serum)	Reported Device Performance (Plasma Imprecision)	Reported Device Performance (Plasma Stability)
Comparability (Plasma vs. Serum)	Regression analysis showing a strong correlation (r close to 1), a slope close to 1, and a small intercept, indicating comparable results between serum and plasma samples.	Site 1: N=45, Range 0.34-24.75 ng/mL, Slope=1.06, Intercept=0.23 ng/mL, r=0.984, Sy.x=1.30 ng/mLSite 2: N=60, Range 0.30-334.20 ng/mL, Slope=1.06, Intercept=0.70 ng/mL, r=0.999, Sy.x=2.45 ng/mLSite 3: N=55, Range 0.64-202.36 ng/mL, Slope=1.06, Intercept=0.56 ng/mL, r=0.995, Sy.x=4.69 ng/mL (Overall: Strong correlation and slopes close to 1 across sites, supporting comparability)	N/A	N/A
Imprecision (Plasma)	Within-run and Total CVs for plasma samples expected to be comparable to established serum performance, indicating good analytical reliability. "No significant difference between the imprecision obtained for serum or plasma" is stated.	N/A	Plasma Samples (N=10, 3 replicates each):- Plasma 1 (Mean 5.61 ng/mL): Within-run CV 2.6%, Total CV 3.2%- Plasma 2 (Mean 6.65 ng/mL): Within-run CV 2.1%, Total CV 2.8%- Plasma 3 (Mean 5.28 ng/mL): Within-run CV 2.9%, Total CV 2.6%- Plasma 4 (Mean 4.48 ng/mL): Within-run CV 2.1%, Total CV 2.4%- Plasma 5 (Mean 6.14 ng/mL): Within-run CV 1.9%, Total CV 2.1%- Plasma 6 (Mean 6.03 ng/mL): Within-run CV 1.4%, Total CV 1.7%- Plasma 7 (Mean 5.23 ng/mL): Within-run CV 2.3%, Total CV 2.3%- Plasma 8 (Mean 4.27 ng/mL): Within-run CV 2.1%, Total CV 2.3%- Plasma 9 (Mean 4.51 ng/mL): Within-run CV 2.3%, Total CV 2.4%- Plasma 10 (Mean 7.27 ng/mL): Within-run CV 2.3%, Total CV 7.4%Serum Controls (for comparison):- Serum Control 1 (Mean 3.31 ng/mL): Within-run CV 3.3%, Total CV 3.7%- Serum Control 2 (Mean 12.75 ng/mL): Within-run CV 1.2%, Total CV 1.2% (Conclusion: Stated there is no significant difference between imprecision for serum and plasma)	N/A
Plasma Sample Stability	Results for refrigerated (2-8°C) and frozen (-20°C) plasma samples should remain consistent with immediate testing over specified periods, demonstrating stability for clinical use. An implied acceptable deviation (e.g., within a certain % or SD) from the baseline.	N/A	N/A	Refrigerated (2-8°C): Samples stable for at least 7 days.Frozen (-20°C): Samples stable for at least 1 month. (Individual results for 10 samples provided, showing minimal variation over time, supporting the stated stability conclusions)

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

Comparability Study (Plasma vs. Serum):
- Sample Size:
  - Site 1: 45 samples
  - Site 2: 60 samples
  - Site 3: 55 samples
  - Total: 160 samples
- Data Provenance: Retrospective and prospective. "Specimens submitted for CK-MB analysis from patients with suspected acute myocardial infarction" (implying clinical samples). "Only specimens from patients who had both serum and lithium heparin plasma samples drawn at the same time were used." The study was conducted at "three independent sites." The country of origin is not specified, but the context generally suggests a developed country (e.g., US or Europe) where such diagnostics are common.
Imprecision Study (Plasma):
- Sample Size: 10 volunteer blood donations, from which plasma was spiked and aliquoted. Each aliquot was tested in three replicates. So, 10 samples, with 3 replicates per measurement point.
- Data Provenance: Prospective (samples from volunteers specifically for the study). Country of origin not specified.
Stability Study (Plasma):
- Sample Size: 10 volunteer blood donations (same as imprecision study).
- Data Provenance: Prospective (samples from volunteers specifically for the study). Country of origin not specified.

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

This type of information is not applicable to this study. The "ground truth" here is the actual concentration of CK-MB in a sample, a quantitative value determined by the Immuno 1 system itself (or implied by its calibration). This is a measurement performance study, not an diagnostic interpretation study by human experts. The comparison baseline for plasma is serum results on the same device.

4. Adjudication Method for the Test Set

This is not applicable. There is no "adjudication" in the sense of resolving discrepancies between human readers or between an AI and human readers. The study involves comparing quantitative measurements from a diagnostic device.

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

This is not applicable. This is an in vitro diagnostic device performance study, not an AI-assisted diagnostic study involving human readers.

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

Yes, the studies described are standalone performance studies of the Immuno 1 CK-MB method. The method itself is an automated laboratory assay (solid-phase enzyme immunoassay). The algorithm used is to "construct the dose response curve" (a quadratic fit through zero) and process results directly from the instrument's optical readings. There is no human interpretation or intervention in the measurement of CK-MB concentration by the algorithm/device. The "performance" being evaluated is the accuracy, precision, and stability of this automated measurement.

7. The Type of Ground Truth Used

The ground truth used in these studies is the quantitative CK-MB concentration as measured by the Immuno 1 system.

For the comparability study, the "ground truth" for plasma is effectively established by the corresponding serum samples run on the same system, assuming serum is the validated or reference matrix. The regression analysis aims to show that plasma measurements are equivalent to serum measurements.
For the imprecision and stability studies, the "ground truth" is the established concentration of the spiked CK-MB in the plasma samples, and the consistency of the device's readings against this established value over time and repeated measurements.

8. The Sample Size for the Training Set

Training Set for the assay's dose response curve: This is built into the assay's calibration process. Calibrators are provided with values of 0, 5, 10, 30, 100, and 300 ng/mL. So, there are 6 calibration points that form the basis for the quadratic fit.
No other "training set" in the context of machine learning (AI model training) is mentioned or relevant here, as this is a traditional immunoassay.

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

The "ground truth" for the calibrators (the training set for the dose-response curve) is established through:

Known concentrations: The calibrators are prepared with specific, pre-defined concentrations of CK-MB (0, 5, 10, 30, 100, and 300 ng/mL). These concentrations would be meticulously prepared and verified using highly accurate reference methods or gravimetric techniques by the manufacturer (Bayer Corp.).
These known concentrations are then used to build the standard curve, allowing the system to convert absorbance measurements into quantitative CK-MB concentrations.

Summary

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Attachment 3

K96 1412

SUMMARY OF SAFETY AND EFFECTIVENESS

CK-MB METHOD FOR THE IMMUNO 1 SYSTEM

Listed below are a comparison of the performance between serum and plasma samples on the Immuno 1 CK-MB method ( T01-3587-51). The information used in this summary of Safety and Effectiveness was extracted from the CK-MB method sheet ( attachment ) and file at Bayer Corp.

The reagents, calibrators and software are the same regardless of whether serum or plasma is used as the sample.

INTENDED USE

ASSAY DESCRIPTION

The assay has a range of 0 to 300 ng/ml and calibrators are provided with values of 0, 5, 10, 30, 100 and 300 ng/ml.

All the results reported herein were obtained by using a quadratic fit through zero algorithm to construct the standard curve.

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ASSAY PERFORMANCE: COMPARISON OF RESULTS WITH SERUM AND PLASMA SAMPLES

The comparison of Immuno 1 CK-MB results with serum and plasma samples was made with specimens submitted for CK-MB analysis from patients with suspected acute myocardial infarction. Only specimens from patients who had both serum and lithium heparin plasma samples drawn at the same time were used. Both serum and plasma samples from a patient were tested together. The study was conducted at three independent sites. The results of the regression analysis is shown in Table 1 below.

Table 1COMPARISON OF IMMUNO 1 CK-MB RESULTS FOR SERUM AND PLASMA.Regression equation isPlasma CK-MB = Serum CK-MB(Slope) + Intercept
	Numberof samples	Range of results(ng/mL)	Slope	Interceptng/mL	r	Sy.xng/mL
Site 1	45	0.34 to 24.75	1.06	0.23	0.984	1.30
Site 2	60	0.30 to 334.20	1.06	0.70	0.999	2.45
Site 3	55	0.64 to 202.36	1.06	0.56	0.995	4.69

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PLASMA SAMPLE HANDLING

A total of 10 volunteers donated about 30 mL of blood each using heparinized vacutainer tubes. The plasma was separated and 10 mL was spiked with about 5 ng/mL of CK-MB. The plasma was then divided into 1mL aliquots. One was assayed immediately after preparation and the others were stored refrigerated or froxen at -20°C. At the intervals indicated in Table 3 below an aliquot was tested on the Immuno 1. The results indicate that a sample can be stored refrigerated for at least 7 days without change in the Immuno 1 result or for one month frozen at -20°C. The results for the first 8 days were obtained using a calibration curve stored at the start of the study. The system was recalibrated for the results with frozen samples after one month storage. The results for the samples are the means of three replicates

Table 2IMMUNO 1 CK-MB RESULTS FOR PLASMA SAMPLES STORED UNDER DIFFERENTCONDITIONS.
	Roomtemperature	Refrigerated at 2 - 8°C					Frozen at-20°C
Sample	Immediate	8 hours	24 hours	48 hours	7 days	8 days	1 month
1	5.6	5.8	5.5	5.5	5.8	5.4	5.6
2	6.7	7.0	6.7	6.5	6.6	6.6	6.3
3	5.2	5.3	5.3	5.3	5.2	5.3	5.2
4	4.4	4.6	4.5	4.5	4.3	4.5	4.2
5	6.1	6.4	6.2	6.1	6.1	6.0	5.8
6	5.9	6.2	6.1	6.0	6.0	6.0	5.7
7	5.1	5.4	5.2	5.3	5.2	5.2	4.8
8	4.2	4.5	4.3	4.3	4.2	4.3	3.9
9	4.4	4.6	4.5	4.6	4.5	4.5	4.3
10	6.8	6.9	7.4	8.2	7.4	7.0	7.3

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IMPRECISION: COMPARISON OF RESULTS WITH SERUM AND PLASMA SAMPLES

Results obtained in the first 8 days of the sample handling study described above were used to calculate the imprecision with plasma samples. This is shown in Table 3. Serum controls had been measured in each run and are included for comparison. There is no significant difference between the imprecision obtained for serum or plasma.

Table 3 : IMPRECISION WITH PLASMA SAMPLES
Sample	Mean(ng/mL)	Within-runSD (ng/mL)	Within-runCV (%)	Total SD(ng/mL)	Total CV(%)
Plasma 1	5.61	0.14	2.6	0.18	3.2
Plasma 2	6.65	0.14	2.1	0.18	2.8
Plasma 3	5.28	0.15	2.9	0.14	2.6
Plasma 4	4.48	0.09	2.1	0.11	2.4
Plasma 5	6.14	0.12	1.9	0.13	2.1
Plasma 6	6.03	0.08	1.4	0.10	1.7
Plasma 7	5.23	0.12	2.3	0.12	2.3
Plasma 8	4.27	0.09	2.1	0.10	2.3
Plasma 9	4.51	0.10	2.3	0.11	2.4
Plasma 10	7.27	0.17	2.3	0.54	7.4
Serum Control 1	3.31	0.11	3.3	0.12	3.7
Serum Control 2	12.75	0.15	1.2	0.28	1.2

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SAMPLE HANDLING INSTRUCTIONS

Serum and plasma (heparin) samples may be used. Samples may be stored for one week at 2 to 8°C or for one month at -20°C. Frozen samples should be thawed at room temperature and mixed thoroughly before use. Thawed samples should not be refrozen. For optimal results the sample must be free of particulate matter.

Increased clotting times may occur with samples from patients receiving anticoagulant or thrombolytic therapy. In those cases only plasma samples should be used! Serum samples containing anticoagulants or thrombolytic agents may vield false positive results on a random basis. Plasma samples collected from these patients using lithium heparin do not appear to exhibit similar problems.

Ensure that clot formation is complete before centrifugation of serum samples. Fibrin may appear in stored plasma samples. All plasma samples should be centrifuged or filtered before analysis to ensure removal of particulate matter.

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Image /page/5/Figure/0 description: The image shows a product label specification for in vitro diagnostics. The label includes the product name "Technicon Immuno 10 CK-MB" and specifies that it contains reagents. The label also includes information such as the product code "T01-3587-51", the net contents "1 x 13.6 mL" and "1 x 6.6 mL", and the discrete number. The label also contains the word "DRAFT" and a signature with the date 11/18/94.

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Attachment 4

Regulation Number and Section

§ 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test system.

(a)
Identification. A creatine phosphokinase/creatine kinase or isoenzymes test system is a device intended to measure the activity of the enzyme creatine phosphokinase or its isoenzymes (a group of enzymes with similar biological activity) in plasma and serum. Measurements of creatine phosphokinase and its isoenzymes are used in the diagnosis and treatment of myocardial infarction and muscle diseases such as progressive, Duchenne-type muscular dystrophy.(b)
Classification. Class II.