MYOGLOBIN ASSAY FOR THE TECHNICON IMMUNO 1 SYSTEM (IN-VITRO DIAGNOSTIC SYSTEM)

K962344 · Bayer Corp. · DDR · Aug 15, 1996 · Immunology

Device Facts

• Record ID: K962344
• Device Name: MYOGLOBIN ASSAY FOR THE TECHNICON IMMUNO 1 SYSTEM (IN-VITRO DIAGNOSTIC SYSTEM)
• Applicant: Bayer Corp.
• Product Code: DDR · Immunology
• Decision Date: Aug 15, 1996
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5680
• Device Class: Class 2

Intended Use

This in vitro diagnostic procedure is a solid phase immunoassay intended for the quantitative determination of Myoglobin in human serum or heparin plasma on the Technicon Immuno 1 system. When used in combination with other clinical data such as presenting symptoms and EKG values, measurement of Myoglobin aides in the early phase diagnosis of Myocardial Infarctions.

Device Story

The Technicon Immuno 1 Myoglobin assay is an in vitro diagnostic enzyme-labeled sandwich immunoassay. It utilizes a monoclonal mouse capture antibody and a polyclonal goat detector antibody. The capture antibody is labeled with fluorescein, and the detector antibody is labeled with alkaline phosphatase (ALP). The solid phase consists of magnetizable particles coated with anti-fluorescein antibodies. Patient serum or heparin plasma samples are mixed with reagents and incubated at 37°C. Myoglobin forms a fluorescein-conjugate/Myoglobin/ALP-conjugate complex, which is captured by the magnetic particles. An external magnetic field precipitates the particles; after washing, p-Nitrophenylphosphate substrate is added. The resulting increase in absorbance is measured spectrophotometrically at 405 and 450 nm. The system calculates results using a Cubic Fit Through Zero dose-response curve. The assay is performed on the Technicon Immuno 1 system in a clinical laboratory setting. Results are used by clinicians alongside EKG and symptom data to assist in the early diagnosis of Myocardial Infarction.

Clinical Evidence

Bench testing only. Performance evaluated via imprecision studies (n=160 replicates), linearity testing, and interference testing (bilirubin, albumin, hemoglobin, gamma globulins, triglycerides, heparin, citrate, urea, creatinine, rheumatoid factor). Method comparison against Behring Nephelometer (n=100 samples) showed a correlation of y = 1.02x + 1.05 (r=0.993). Sensitivity (minimum detectable concentration) is 1.8 ng/mL. Normal distribution established using 77 non-AMI individuals.

Technological Characteristics

Solid-phase enzyme-labeled sandwich immunoassay. Components: monoclonal mouse capture antibody (fluorescein-labeled), polyclonal goat detector antibody (ALP-labeled), magnetizable particles coated with anti-fluorescein. Energy source: spectrophotometric measurement at 405/450 nm. Assay range: 0-3000 ng/mL. Calibration: Cubic Fit Through Zero. System: Technicon Immuno 1.

Indications for Use

Indicated for the quantitative determination of Myoglobin in human serum or heparin plasma to aid in the early diagnosis of Myocardial Infarction in patients presenting with symptoms suggestive of cardiac events.

Regulatory Classification

Identification

A myoglobin immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the myoglobin (an oxygen storage protein found in muscle) in serum and other body fluids. Measurement of myoglobin aids in the rapid diagnosis of heart or renal disease.

Predicate Devices

• Behring N Latex Myoglobin Reagents

Submission Summary (Full Text)

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Attachment 3

# AUG 15 1996

# Summary of Safety and Effectiveness

K962344

# MYOGLOBIN METHOD FOR THE IMMUNO 1 SYSTEM

Listed below is a comparison of the performance between the Immuno 1 Myoglobin method (T01-3653-51) and a similar device that was granted FDA determination of substantial equivalence: The Behring N Latex Myoglobin Reagents. This reagents are designed to run on the Behring Nephelometer. The information used in this summary of Safety and Effectiveness was extracted from the Myoglobin Method Sheet and from data on file at Bayer Corporation.

# Intended Use

This in vitro diagnostic procedure is a solid phase immunoassay intended for the quantitative determination of Myoglobin in human serum or heparin plasma on the Technicon Immuno 1 system. When used in combination with other clinical data such as presenting symptoms and EKG values, measurement of Myoglobin aides in the early phase diagnosis of Myocardial Infarctions.

# Assay Description

The method described is an enzyme label sandwich assay using a monoclonal (mouse) capture and a polyclonal (goat) detector antibody. The monoclonal antibody is labelled with fluorescein and the polyclonal antibody labelled with alkaline phosphatase (ALP). The two reagents are the active compounds of the R1 and the R2 reagent, respectively. The solid phase consists of a suspension of magnetizable particles coated with antibody to fluorescein (mIMP reagent). Sample or calibrator, R1 and R2 reagent and mIMP reagent are mixed simultaneously and incubated at $37^{\circ}\mathrm{C}$ . In the presence of Myoglobin a fluorescein-conjugate $\equiv$ Myoglobin $\equiv$ ALP-conjugate complex is formed and captured by the antiFluorescein antibodies on the magnetic particles. The particles are precipitated by an external magnetic field, washed and paraNitrophenylphosphate is added as the enzyme substrate. The increase in absorbance due to the formation of p-Nitrophenolate is monitored spectrophotometrically at 405 and $450\mathrm{nm}$ . The response read is directly proportional to the

Final result after 23 minutes
Fig. 1 Schematic representation of the Technicon Immuno 1 Myoglobin Assay

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concentration of Myoglobin in a sample. A Cubic Fit Through Zero is used to calculate the dose response curve. The assay is depicted schematically in fig. 1.

The assay has a range of 0 to 3000 ng/mL with a sensitivity of 1.8 ng/mL; six calibrators with Myoglobin concentrations of 0, 60, 180, 600, 1500 and 3000 ng/mL are provided.

A dose response curve is shown in fig. 2.

Fig. 2 Calibration Curve of the Technicon Immuno 1 Myoglobin Assay

Data pairs measured:

0 ng/mL: 0.0019 AU/min
60 ng/mL: 0.0645 AU/min
180 ng/mL: 0.1937 AU/min
600 ng/mL: 0.6805 AU/min
1500 ng/mL: 1.7656 AU/min
3000 ng/mL: 3.1391 AU/min

# ASSAY PERFORMANCE

## Imprecision

Total imprecision data was obtained by analyzing human serum controls on two Immuno 1 instruments on 20 different days. Two separate lots of reagents and calibrators were used. Both reagent and calibrator combinations on both instruments were tested with two different lots of magnetic particles. The total number of replicates for each level was 160. The calibration was only performed when a new reagent/ calibrator lot or particle lot combination was implemented on a machine.

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| Table 1: Imprecision of Immuno 1 Myoglobin Assay Data was collected on two Systems over twenty days with four replicates on each day on each system | | | | | |
| --- | --- | --- | --- | --- | --- |
| Specimen | Total Imprecision (n= 160) | | | Within run imprecision (mean) | |
| | Average [ng/mL] | Std Dev [ng/mL] | CV [%] | Std Dev [ng/mL] | CV [%] |
| Sample 1 | 14.8 | 0.8196 | 5.5 | 0.3081 | 2.1 |
| Sample 2 | 52.6 | 1.9084 | 3.6 | 0.77925 | 1.5 |
| Sample 3 | 75.6 | 2.9831 | 3.9 | 1.06855 | 1.4 |
| Sample 4 | 131.3 | 4.6636 | 3.6 | 2.28195 | 1.7 |
| Sample 5 | 247 | 10.9436 | 4.4 | 4.47045 | 1.8 |
| Sample 6 | 278.1 | 8.2182 | 3.0 | 3.53275 | 1.3 |
| Sample 7 | 639.7 | 23.0701 | 3.6 | 11.3425 | 1.8 |
| Sample 8 | 1557.6 | 56.46 | 3.6 | 21.4224 | 1.4 |
| Sample 9 | 2718.9 | 91.3937 | 3.4 | 44.90395 | 1.7 |

Correlation with Immuno 1 Myoglobin results with Behring Nephelometer A

A total of 100 serum and plasma samples with Behring Nephelometer (BNA) values in the range of 21 to 2660 ng/mL (BNA) were tested with the Behring Nephelometer A and the Immuno 1 Myoglobin assay. The correlation equation according to Bablock-Passing was

$$
y = 1.02 \times x + 1.05
$$

(y is Immuno 1 Myoglobin assay; x is Behring Nephelometer A Myoglobin assay).

| Calculation of Regression Line : | | | |
| --- | --- | --- | --- |
| Slope (b) : | 1.02 | | Number of Samples : 100 |
| Limits : | 0.98 | 1.06 | Sampletype : all sample codes |
| Intercept (a) : | 1.05 | | |
| Limits : | -3.2 | 4.5 | |
| Confidence of Correlation : | 0.99314 | | |

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Fig. 3 Correlation Plot of Immuno 1 Myoglobin Results versus Behring Nephelometer A results

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31-JUL-1996 16:42
BAYER AG , ABT. ZF-F
[phone] S.02
TEST.XLG (METHOD.XLD)

# Method Comparison acc. to Bablok-Passing

Methods: Immuno 1 Myoglobin vs. Behring N-Latex Reagents (BNA)

Serum and Plasma Samples

♦ Values
— Regression Line
— Identity Line

Calculation of Regression Line:

| Slope (b) : | 1.04110 | Number of Samples : | 54 |
| --- | --- | --- | --- |
| Limits : | 0.96763 | 1.12364 | Sampletype : all sample codes |
| Intercept (a) : | -0.98356 | | |
| Limits : | -5.79055 | 4.21007 | |
| Confidence of Correlati- | 0.96814 | | |

Bayer Leverkusen, Dr. Petry
4a
PEY 31.07.1996

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# Interference

For all interference measurements a +2 Pool and a -2 Pool was prepared. The +2 Pool was made from a solution of Myoglobin in serum with a concentration of approximately 180 µg/l (double of the concentration at the medical decision point) being diluted 1+1 with a solution of the potentially interfering substance in Myoglobin-stripped serum at a concentration twice as high as required. This yields a Myoglobin concentration at the medical decision level with the required interferant-concentration. The -2 Pool was made up from the same Myoglobin solution, but this time being diluted 1+1 with stripped serum containing no interferant. So two solutions of exactly identical Myoglobin concentration were obtained, one containing no interferent, the other with the required high concentration. The 0-Pool was obtained mixing equivalent amounts of the +2- and the -2-Pool while the -1- and +1-Pool were prepared from equal quantities of the 0-Pool and the -2- respectively the +2-Pool.

## - Bilirubin

Bilirubin:
- -2 Pool: 0 mg/dL
- +2 Pool: 25 mg/dL

Results:

| Pool | conc. (meas.) | % of -2 pool |
| --- | --- | --- |
| 2- | 90.6 | 100.0 |
| 1- | 90.6 | 100.0 |
| 0 | 91.3 | 100.8 |
| 1+ | 91.5 | 101 |
| 2+ | 90.9 | 100.3 |

## - Albumin

Albumin:
- -2 Pool: 0 mg/mL
- +2 Pool: 6.5 g/dL

Results:

| Pool | conc (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 87.1 | 100.0 |
| 1- | 87.1 | 100.0 |
| 0 | 87.9 | 100.9 |
| 1+ | 88.1 | 101.1 |
| 2+ | 88.4 | 101.5 |

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# - Hemoglobin

Hemoglobin: -2 Pool: 0 mg/mL
+2 Pool: 1 g/dL

Results:
| Pool | conc. (meas) | % of -2 Pool |
| --- | --- | --- |
| 2- | 88 | 100.0 |
| 1- | 89.3 | 101.5 |
| 0 | 89.8 | 102 |
| 1+ | 88.1 | 100.1 |
| 2+ | 89.3 | 101.5 |

# - Gamma Globulins

BGG: -2 Pool: 0 mg/dL
+2 Pool: 5.3 g/dL

Results:
| Pool | conc. (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 82,7 | 100,0 |
| 1- | 84,1 | 101,7 |
| 0 | 84,6 | 102,3 |
| 1+ | 78,6 | 95 |
| 2+ | 85,6 | 103.5 |

# - Triglycerides

Triglyceride Supertrate: -2 Pool: 0 g/dL
+2 Pool: 1.3 g/dL (calculated for Triglycerides)

Results:
| Pool | conc. (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 94,9 | 100,0 |
| 1- | 90,4 | 95,3 |
| 0 | 90 | 94.8 |
| 1+ | 89 | 93.8 |
| 2+ | 99.2 | 104.5 |

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# - Heparin

Heparin: -2 Pool: 0 IU/mL (Serum)
+2 Pool: 65 IU/mL (0.5 mg/mL)

Results:

| Pools | conc. (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 85,9 | 100,0 |
| 1- | 85,8 | 99,9 |
| 0 | 85,8 | 99,9 |
| 1+ | 86,4 | 100,6 |
| 2+ | 87,2 | 101,5 |

# - Citrate

Trisodium Citrate, Dihydrate: -2 Pool: 0 mg/ml
+2Pool: 50 mg/mL

Results:

| Pools | conc. (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 92,4 | 100,0 |
| 1- | 90,8 | 98,3 |
| 0 | 91,5 | 99 |
| 1+ | 91,1 | 98,6 |
| 2+ | 88,7 | 96 |

# - Urea and Creatine

Urea and Creatine: -2 Pool: no Urea, no Creatine
+2 Pool: 200 mg/dL Urea, 2.5 mg/dL Creatine

Results:

| Pools | conc. (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 127,7 | 100,0 |
| 1- | 129,2 | 101,2 |
| 0 | 127,9 | 100,2 |
| 1+ | 127,5 | 99,8 |
| 2+ | 128,4 | 100,5 |

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# - Rheumatory Factor

Rheumatory Factor: -2 Pool: 0 IU/mL
+2 Pool: 567 IU/mL

## Results:

| Pool | conc. (meas.) | % of -2 Pool |
| --- | --- | --- |
| 2- | 95,9 | 100,0 |
| 1- | 96,1 | 100,2 |
| 0 | 98,1 | 102,3 |
| 1+ | 98,8 | 103 |
| 2+ | 99,6 | 103,9 |

## Linearity

All control to check linearity pools were generated in a way, that a serum sample with a high Myoglobin level (+2 Pool) and a low sample (-2 Pool) were mixed in a ratio of 1 + 1. The 0 Pool thus generated was furtherly mixed with the same amount of the +2 respectively the -2 Pool to have the +1 and -1 Pool. By that procedure five equally spaced controls covering the whole assay range are generated. For data analysis a linear regression was calculated from the result of the -2, -1 and 0 Pools of each sample series. The expected results and the deviation from the measured values were calculated from the equation. The result is shown in the table below.

| Pool (Sample) | measured | calculated | dev (%) |
| --- | --- | --- | --- |
| -2 (A) | 18.7 | 19.5 | -3.9 |
| -1 (A) | 656.7 | 655.2 | 0.2 |
| 0 (A) | 1290.2 | 1291 | -0.1 |
| 1 (A) | 1855.7 | 1926.7 | -3.7 |
| 2 (A) | 2481.8 | 2562.5 | -3.1 |
| -2 (B) | 19.1 | 24.2 | -21 |
| -1 (B) | 659.9 | 649.7 | 1.6 |
| 0 (B) | 1270.2 | 1275.3 | -0.4 |
| 1 (B) | 1862.7 | 1900.8 | -2 |
| 2 (B) | 2439.2 | 2526.4 | -3.5 |
| -2 (C) | 19.1 | 20.4 | -6.5 |
| -1 (C) | 664.9 | 662.2 | 0.4 |
| 0 (C) | 1302.7 | 1304 | -0.1 |
| 1 (C) | 1891.6 | 1945.8 | -2.8 |
| 2 (C) | 2510.9 | 2587.6 | -3 |

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# Sample Dilution

For testing Sample Dilution of clinical serum and plasma samples a dilution series with Immuno 1 Sample Diluent B and Immuno 1 Myoglobin Calibrator Level 1 was run. The recovery of the undiluted sample was set to 100%

| Dilution of Clinical Serum Samples with Immuno 1 Sample Diluent B | | | | | |
| --- | --- | --- | --- | --- | --- |
| sample content % | Identification | AU/min | conc.(meas.) | conc.(calc) | recovery (%) |
| 100 | Serum Sample A | 1.1004 | 880.5 | 880.5 | 100 |
| 75 | | 0.7783 | 623.3 | 660.4 | 106.0 |
| 50 | | 0.4671 | 426.6* | 440.3 | 103.2 |
| 25 | | 0.2536 | 205.1 | 220.1 | 107.3 |
| 10 | | 0.1013 | 81.6 | 88.1 | 108 |
| 0 | | 0.0027 | 0.6 | 0.0 | - |
| 100 | Serum Sample B | 2.7817 | 2420.1 | 2420.1 | 100 |
| 75 | | 2.2541 | 1881.2 | 1815.1 | 96.5 |
| 50 | | 1.5296 | 1233 | 1210.1 | 98.1 |
| 25 | | 0.7516 | 602.1 | 605.0 | 100.5 |
| 10 | | 0.2922 | 236.2 | 242.0 | 102.5 |
| 0 | | 0.002 | 0.1 | 0.0 | - |
| 100 | Serum Sample C | 3.1881 | 2887.9 | 2887.9 | 100 |
| 75 | | 2.5717 | 2197.4 | 2165.9 | 98.6 |
| 50 | | 1.7897 | 1456.1 | 1444.0 | 99.2 |
| 25 | | 0.9565 | 765.2 | 722.0 | 94.4 |
| 10 | | 0.3813 | 307.7 | 288.8 | 93.9 |
| 0 | | 0.0021 | 0.2 | 0.0 | - |
| 100 | Serum Sample D | 2.7268 | 2360.9 | 2360.9 | 100 |
| 75 | | 2.0877 | 1724.3 | 1770.7 | 102.7 |
| 50 | | 1.4969 | 1205.5 | 1180.5 | 97.9 |
| 25 | | 0.7662 | 613.7 | 590.2 | 96.2 |
| 10 | | 0.2915 | 235.6 | 236.1 | 100.2 |
| 0 | | 0.0023 | 0.3 | 0.0 | - |
| 100 | Serum Sample E | 2.6725 | 2302.7 | 2302.7 | 100 |
| 75 | | 2.0673 | 1705.4 | 1727.0 | 101.3 |
| 50 | | 1.456 | 1171.4 | 1151.4 | 98.3 |
| 25 | | 0.7215 | 578.2 | 575.7 | 99.6 |
| 10 | | 0.2689 | 217.5 | 230.3 | 105.9 |
| 0 | | 0.002 | 0.1 | 0.0 | - |

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| Dilution of Serum Samples with Myoglobin Calibrator Level 1 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample content % | Identification | AU/min | conc. (meas.) | conc. (calc) | dev. (%) |
| 100 | Serum Sample A | 1.0525 | 859.7 | 859.7 | 100 |
| 75 | | 0.8022 | 659.3 | 644.8 | 97.8 |
| 50 | | 0.5132 | 426.3 | 429.9 | 100.8 |
| 25 | | 0.2648 | 222.1 | 214.9 | 96.8 |
| 10 | | 0.0976 | 81 | 86.0 | 106.2 |
| 0 | | 0.0019 | -1.3 | 0.0 | - |
| 100 | Serum Sample B | 2.7167 | 2344.3 | 2344.3 | 100 |
| 75 | | 2.1286 | 1767.2 | 1758.2 | 99.5 |
| 50 | | 1.4933 | 1217.3 | 1172.2 | 96.3 |
| 25 | | 0.7267 | 598.8 | 586.1 | 97.9 |
| 10 | | 0.2955 | 247.5 | 234.4 | 94.7 |
| 0 | | 0.003 | -0.3 | 0.0 | - |
| 100 | Serum Sample C | 3.0948 | 2765.1 | 2765.1 | 100 |
| 75 | | 2.4005 | 2023.7 | 2073.8 | 102.5 |
| 50 | | 1.7318 | 1417.3 | 1382.6 | 97.6 |
| 25 | | 0.9207 | 754.1 | 691.3 | 91.7 |
| 10 | | 0.3701 | 309.3 | 276.5 | 89.4 |
| 0 | | 0.0018 | -1.4 | 0.0 | - |
| 100 | Serum Sample D | 2.8607 | 2499.2 | 2499.2 | 100 |
| 75 | | 2.1077 | 1748.1 | 1874.4 | 107.2 |
| 50 | | 1.4938 | 1217.7 | 1249.6 | 102.6 |
| 25 | | 0.751 | 618.3 | 624.8 | 101.1 |
| 10 | | 0.2835 | 237.7 | 249.9 | 105.1 |
| 0 | | 0.0019 | -1.3 | 0.0 | - |
| 100 | Serum Sample E | 2.7182 | 2345.6 | 2345.6 | 100 |
| 75 | | 2.1273 | 1766 | 1759.2 | 99.6 |
| 50 | | 1.4916 | 1215.9 | 1172.8 | 96.5 |
| 25 | | 0.7647 | 629.3 | 586.4 | 93.2 |
| 10 | | 0.297 | 248.8 | 234.6 | 94.3 |
| 0 | | 0.0021 | -1.1 | 0.0 | - |
| Dilution of Plasma Samples with Immuno 1 Sample diluent B | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample content [%] | Identification | AU/min | conc. (meas) | conc (calc) | dev (%) |
| 100 | Plasma Sample AA | 1.44 | 1224.1 | 1224.1 | 100 |
| 75 | | 1.0586 | 898 | 918.075 | 102.2 |

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| Sample content [%] | Identification | AU/min | conc. (meas) | conc (calc) | dev (%) |
| --- | --- | --- | --- | --- | --- |
| 50 | | 0.6935 | 591.2 | 612.05 | 103.5 |
| 25 | | 0.3398 | 292.3 | 306.025 | 104.7 |
| 10 | | 0.1379 | 118.7 | 122.41 | 103.1 |
| 0 | | 0.0024 | 0.1 | 0 | - |
| 100 | Plasma Sample BB | 2.6022 | 2332.7 | 2332.7 | 100 |
| 75 | | 2.0189 | 1747.1 | 1749.525 | 100.1 |
| 50 | | 1.3546 | 1150.3 | 1166.35 | 101.4 |
| 25 | | 0.6843 | 583.5 | 583.175 | 99.9 |
| 10 | | 0.2568 | 221.3 | 233.27 | 105.4 |
| 0 | | 0.0023 | -0.1 | 0 | - |
| 100 | Plasma Sample CC | 2.5736 | 2302.1 | 2302.1 | 100 |
| 75 | | 1.9753 | 1705.9 | 1726.575 | 101.2 |
| 50 | | 1.361 | 1155.8 | 1151.05 | 99.6 |
| 25 | | 0.6662 | 568.3 | 575.525 | 101.3 |
| 10 | | 0.2714 | 233.9 | 230.21 | 98.4 |
| 0 | | 0.0022 | -0.2 | 0 | - |
| 100 | Plasma Sample DD | 1.2854 | 1090.7 | 1090.7 | 100 |
| 75 | | 0.9627 | 817.3 | 818.025 | 100.1 |
| 50 | | 0.635 | 542.1 | 545.35 | 100.6 |
| 25 | | 0.3104 | 267.3 | 272.675 | 102 |
| 10 | | 0.1269 | 109.1 | 109.07 | 100 |
| 0 | | 0.002 | -0.4 | 0 | - |
| 100 | Plasma Sample EE | 1.62 | 1382.2 | 1382.2 | 100 |
| 75 | | 1.2144 | 1030.1 | 1036.65 | 100.6 |
| 50 | | 0.8063 | 685.9 | 691.1 | 100.8 |
| 25 | | 0.3932 | 337.8 | 345.55 | 102.3 |
| 10 | | 0.1604 | 138.2 | 138.22 | 100 |
| 0 | | 0.0022 | -0.1 | 0 | - |
| Dilution of Plasma Samples with Immuno 1 Calibrator Level 1 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample content % | Identification | AU/min | conc (meas) | conc (calc) | dev (%) |
| 100 | Plasma Sample AA | 1.4613 | 1216.1 | 1216.1 | 100 |
| 75 | | 1.1144 | 929.1 | 912.075 | 98.2 |
| 50 | | 0.814 | 683.6 | 608.05 | 88.9 |
| 25 | | 0.3588 | 307 | 304.025 | 99 |
| 10 | | 0.1268 | 108.8 | 121.61 | 111.8 |
| 0 | | 0.0025 | -0.1 | 0 | - |

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| Dilution of Plasma Samples with Immuno 1 Calibrator Level 1 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample content % | Identification | AU/min | conc (meas) | conc (calc) | dev (%) |
| 100 | Plasma Sample BB | 2.724 | 2392.9 | 2392.9 | 100 |
| 75 | | 2.1735 | 1843.6 | 1794.675 | 97.3 |
| 50 | | 1.4542 | 1210.1 | 1196.45 | 98.9 |
| 25 | | 0.7596 | 639.2 | 598.225 | 93.6 |
| 10 | | 0.283 | 242.9 | 239.29 | 98.5 |
| 0 | | 0.0023 | -0.2 | 0 | - |
| 100 | Plasma Sample CC | 2.7637 | 2435.5 | 2435.5 | 100 |
| 75 | | 2.1392 | 1811.5 | 1826.625 | 100.8 |
| 50 | | 1.4943 | 1243.9 | 1217.75 | 97.9 |
| 25 | | 0.7273 | 612.7 | 608.875 | 99.4 |
| 10 | | 0.2993 | 256.7 | 243.55 | 94.9 |
| 0 | | 0.0023 | -0.3 | 0 | - |
| 100 | Plasma Sample DD | 1.3432 | 1117.6 | 1117.6 | 100 |
| 75 | | 1.0524 | 878.4 | 838.2 | 95.4 |
| 50 | | 0.6883 | 580.7 | 558.8 | 96.2 |
| 25 | | 0.3372 | 288.8 | 279.4 | 96.7 |
| 10 | | 0.1365 | 117.3 | 111.76 | 95.3 |
| 0 | | 0.0021 | -0.4 | 0 | - |
| 100 | Plasma Sample EE | 1.6146 | 1345.7 | 1345.7 | 100 |
| 75 | | 1.2409 | 1033 | 1009.275 | 97.7 |
| 50 | | 0.8323 | 698.6 | 672.85 | 96.3 |
| 25 | | 0.3987 | 340.5 | 336.425 | 98.8 |
| 10 | | 0.1619 | 139.1 | 134.57 | 96.7 |
| 0 | | 0.0021 | -0.4 | 0 | - |

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# Hook Effect

Samples with Myoglobin concentrations up to 1 Million ng/mL were assayed with the Technicon Immuno 1 Myoglobin Assay. The assay will not erroneously compute raw data to concentrations within the calibration range of the assay as long as the Myoglobin content in the sample is less than 150,000 ng/mL. In the following figure there is a graphical representation of the expected concentrations against the reported for antigen levels between 500 ng/mL and 1 Million ng/mL.

Fig. 4 Concentrations reported by the Immuno 1 Myoglobin assay against expected concentrations. There are no results reported if they above the dashed line in Fig. 4.

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# Recovery

Known amounts of Myoglobin Solution were spiked into four clinical samples, two serum samples, two plasma samples. For serum the recoveries were ranging from 96.5 to 105.2%; for plasma the recoveries are between 97.7 and 103%. For the recoveries of spiked antigen in plasma it is important to also use Myoglobin containing plasma as the spiking material. Serum- of buffer-based material may lead to deviations from the expected.

| Serum (PEY 2795) | | | |
| --- | --- | --- | --- |
| Sample | Expected | found | recovery |
| Serum
Sample α | 39.1 | 39.1 | 100.0 |
| | 77.2 | 78.1 | 101.2 |
| | 651 | 672.6 | 103.3 |
| | 1224 | 1250.1 | 102.1 |
| | 1798 | 1757.7 | 97.8 |
| | 2371 | 2377.2 | 100.3 |
| Serum
Sample β | 38.5 | 38.5 | 100.0 |
| | 77.2 | 77.4 | 100.3 |
| | 651 | 685 | 105.2 |
| | 1224 | 1223.6 | 100.0 |
| | 1798 | 1735.1 | 96.5 |
| | 2371 | 2331.8 | 98.3 |
| Plasma (PEY 2795) | | | |
| --- | --- | --- | --- |
| Sample | expected | found | recovery |
| Plasma
Sample γ | 24.8 | 24.8 | 100.0 |
| | 77 | 78.1 | 101.4 |
| | 843.45 | 860.6 | 102.0 |
| | 1609.9 | 1624.3 | 100.9 |
| | 2376.35 | 2326.6 | 97.9 |
| | 3142.8 | 3180.4 | 101.2 |
| Plasma
Sample δ | 31.3 | 31.3 | 100.0 |
| | 83.5 | 81.6 | 97.7 |
| | 758.45 | 780.9 | 103.0 |
| | 1433.4 | 1459.2 | 101.8 |
| | 2108.35 | 2078 | 98.6 |
| | 2783.3 | 2782 | 100.0 |

{15}

# Expected Values

Samples from 77 non AMI individuals were assayed and gave the distribution of results shown in Fig. 4.

It was found that 98% of the values were 88 ng/mL or less.

Immuno 1 Myoglobin: Normal Distribution
Fig. 5 Technicon Immuno 1 Myoglobin Assay: Normal Distribution

# Minimum Detectable Concentration

The minimum detectable concentration was measured in 32 different runs on four different days using two different lots of reagents, calibrators and magnetic particles. The L1 calibrator containing no Myoglobin was measured 576 times all together. Calculated from the mean zero absorption plus two standard deviations the minimum detectable dose was determined as 1.8 ng/mL.