CK-MB METHOD FOR THE TECHNICON IMMUNO 1 SYSTEM IN-VITRO

K961412 · Bayer Corp. · JHY · Jun 18, 1996 · Clinical Chemistry

Device Facts

• Record ID: K961412
• Device Name: CK-MB METHOD FOR THE TECHNICON IMMUNO 1 SYSTEM IN-VITRO
• Applicant: Bayer Corp.
• Product Code: JHY · Clinical Chemistry
• Decision Date: Jun 18, 1996
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1215
• Device Class: Class 2

Intended Use

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 Story

Immuno 1 CK-MB is a solid-phase enzyme immunoassay for quantitative CK-MB measurement in human serum or plasma. System uses two monoclonal antibodies: one CK-MB specific (fluorescein-labeled) and one B-subunit specific (alkaline phosphatase-labeled). Magnetizable particles coated with anti-fluorescein capture the complex. ALP activity measured via pNPP substrate conversion to para-nitrophenoxide; absorbance monitored at 405nm and 450nm. Used in clinical laboratories; operated by technicians. Results aid physicians in diagnosing acute myocardial infarction when combined with clinical data and EKG values. Enables rapid assessment of cardiac injury.

Clinical Evidence

Bench testing only. Comparison study across three independent sites using 160 paired serum and lithium heparin plasma samples from patients with suspected acute myocardial infarction. Regression analysis showed high correlation (r = 0.984 to 0.999). Imprecision study (n=10 volunteers) evaluated storage conditions (refrigerated 7 days, frozen 1 month) and within-run/total CVs, showing no significant difference between serum and plasma performance.

Technological Characteristics

Solid-phase enzyme immunoassay; magnetizable particle separation; alkaline phosphatase (ALP) label; pNPP substrate; dual-wavelength absorbance detection (405nm/450nm); quadratic fit through zero algorithm; compatible with Technicon Immuno 1 system.

Indications for Use

Indicated for quantitative determination of CK-MB in human serum or plasma to aid in the diagnosis of acute myocardial infarction in patients presenting with symptoms or EKG values suggestive of cardiac events.

Regulatory Classification

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.

Submission Summary (Full Text)

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Attachment 3
K961412

# 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 from data on 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

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.

## ASSAY 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 $37^{\circ}\mathrm{C}$. 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 $405\mathrm{nm}$ and $450\mathrm{nm}$. 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 $300\mathrm{ng / ml}$ and calibrators are provided with values of 0, 5, 10, 30, 100 and $300\mathrm{ng / ml}$.

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 1
COMPARISON OF IMMUNO 1 CK-MB RESULTS FOR SERUM AND PLASMA.
Regression equation is
Plasma CK-MB = Serum CK-MB(Slope) + Intercept | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Number
of samples | Range of results
(ng/mL) | Slope | Intercept
ng/mL | r | Sy.x
ng/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 1 mL aliquots. One was assayed immediately after preparation and the others were stored refrigerated or frozen 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 2
IMMUNO 1 CK-MB RESULTS FOR PLASMA SAMPLES STORED UNDER DIFFERENT CONDITIONS. | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Room temperature | 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-run SD (ng/mL) | Within-run CV (%) | 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 yield 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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Attachment 4

# IN VITRO DIAGNOSTIC PRODUCT LABEL SPECIFICATION


PROOF IS AT: 200%
SIZE: 1.875" x 2.5"

COLORS
☐ BLACK

REPRODUCTION TYPE SIZE:
BODY COPY: 8 pt
DISCRETE NO: 6 pt

PRELIMINARY

11/18/94

DRAFT