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 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, R1and R2 reagent and mIMP reagent are mixed simultaneously and incubated at 37 °C. In the presence of Myoglobin fluorescein-conjugate= Myoglobin=ALPa 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 para-Nitrophenylphosphate is added as the enzyme substrate. The increase in absorbance due to the formation of p-Nitrophenolate is monitored spectrophotometrically at 405 and 450 nm. The 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.

AI/ML Overview

This document describes the performance characteristics of the MYOGLOBIN METHOD FOR THE IMMUNO 1 SYSTEM.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical targets in the provided text. Instead, the document presents performance data to demonstrate the device's characteristics. The reported performance metrics are summarized below:

Performance Characteristic	Reported Device Performance
Imprecision (Total CV)
Sample 1 (14.8 ng/mL)	5.5 %
Sample 2 (52.6 ng/mL)	3.6 %
Sample 3 (75.6 ng/mL)	3.9 %
Sample 4 (131.3 ng/mL)	3.6 %
Sample 5 (247 ng/mL)	4.4 %
Sample 6 (278.1 ng/mL)	3.0 %
Sample 7 (639.7 ng/mL)	3.6 %
Sample 8 (1557.6 ng/mL)	3.6 %
Sample 9 (2718.9 ng/mL)	3.4 %
Correlation with Behring Nephelometer A
Correlation Equation (y = Immuno 1, x = BNA)	y = 1.02 × x + 1.05
Slope (b)	1.02 (Limits: 0.98 - 1.06)
Intercept (a)	1.05 (Limits: -3.2 - 4.5)
Confidence of Correlation	0.99314
Interference (measured concentration as % of -2 pool)
Bilirubin (25 mg/dL)	100.3% - 101%
Albumin (6.5 g/dL)	100.9% - 101.5%
Hemoglobin (1 g/dL)	100.1% - 102%
Gamma Globulins (5.3 g/dL)	78.6% - 103.5% (The result for 1+ (95) and 2+ (103.5) are presented in a confusing manner, but the measured concentrations are within a reasonable range of the -2 pool)
Triglycerides (supertrate)	93.8% - 104.5%
Heparin (65 IU/mL)	99.9% - 101.5%
Citrate (50 mg/mL)	96% - 99%
Urea and Creatine (200 mg/dL Urea, 2.5 mg/dL Creatine)	99.8% - 101.2%
Rheumatoid Factor (567 IU/mL)	100.2% - 103.9%
Linearity (deviation from calculated)
Range	-21% to 1.6%
Sample Dilution (Recovery)
Serum samples dilated with Immuno 1 Sample Diluent B	93.9% - 108%
Serum samples dilated with Immuno 1 Calibrator Level 1	93.2% - 107.2%
Plasma samples dilated with Immuno 1 Sample Diluent B	99.9% - 105.4%
Plasma samples diluted with Immuno 1 Calibrator Level 1	88.9% - 111.8%
Hook Effect	No erroneous results within the calibration range for Myoglobin content < 150,000 ng/mL.
Recovery of spiked samples
Serum samples	96.5% - 105.2%
Plasma samples	97.7% - 103%
Expected Values (Normal Distribution)	98% of values were 88 ng/mL or less for non-AMI individuals (n=77).
Minimum Detectable Concentration	1.8 ng/mL
Assay Range	0 to 3000 ng/mL
Sensitivity	1.8 ng/mL

2. Sample sizes used for the test set and the data provenance

Imprecision:
- Sample Size: 160 replicates for each of the 9 samples/levels tested (total of 1440 measurements across two instruments, 20 days, 4 replicates/day/system).
- Data Provenance: Not explicitly stated, but the "human serum controls" suggest a clinical laboratory setting. It is not specified if these were retrospective or prospective.
Correlation with Behring Nephelometer A:
- Sample Size: 100 serum and plasma samples.
- Data Provenance: Not explicitly stated, but the use of "serum and plasma samples" implies clinical samples. It is not specified if these were retrospective or prospective.
Interference:
- Sample Size: For each interferent, multiple "pools" (-2, -1, 0, +1, +2) were prepared and measured. The exact number of replicates for each pool is not specified, but the setup implies multiple measurements for each.
- Data Provenance: "Myoglobin in serum" and "Myoglobin-stripped serum" with added interfering substances indicates a laboratory-controlled study.
Linearity:
- Sample Size: Not explicitly stated, but the creation of five equally spaced controls (-2, -1, 0, +1, +2) for three different series (A, B, C) suggests multiple measurements. For data analysis, linear regression was calculated from the -2, -1 and 0 Pools of each series.
- Data Provenance: "Serum sample with a high Myoglobin level (+2 Pool) and a low sample (-2 Pool)" indicates laboratory-controlled experiments.
Sample Dilution:
- Sample Size: Multiple dilution series were performed for both serum (5 samples tested with 2 diluent types) and plasma (5 samples tested with 2 diluent types). Each series involved 6 different dilution points (100%, 75%, 50%, 25%, 10%, 0%). This is a total of 10 samples for serum and 10 samples for plasma being tested across different dilutions with two different diluents, meaning 20 samples in total tested across 6 dilution points.
- Data Provenance: "Clinical serum and plasma samples" were used, suggesting human origin. It's not specified if these were retrospective or prospective.
Hook Effect:
- Sample Size: Not explicitly stated, but "Samples with Myoglobin concentrations up to 1 Million ng/mL were assayed." The figure shows data points from 500 ng/mL to 1 Million ng/mL.
- Data Provenance: Laboratory-controlled samples.
Recovery:
- Sample Size: Four clinical samples (two serum, two plasma). Each sample was spiked at multiple levels (6 different concentrations, including the unspiked sample).
- Data Provenance: "Clinical samples" suggest human origin. Not specified if retrospective or prospective.
Expected Values:
- Sample Size: 77 non-AMI individuals.
- Data Provenance: "Samples from 77 non AMI individuals." This indicates human clinical samples. It's not specified if these were retrospective or prospective, or their country of origin.
Minimum Detectable Concentration:
- Sample Size: The L1 calibrator (0 Myoglobin) was measured 576 times.
- Data Provenance: Laboratory-controlled measurements.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

This document describes an in vitro diagnostic assay. For such devices, "ground truth" typically refers to established analytical methods or reference values for the analytes being measured, or clinical diagnoses for determining clinical performance (e.g., AMI status).

For analytical performance (Imprecision, Correlation, Interference, Linearity, Sample Dilution, Hook Effect, Recovery, Minimum Detectable Concentration): The ground truth is intrinsically defined by the analytical methods used (e.g., mass spectrometry, existing validated assays like the Behring Nephelometer A), and the precise concentrations of prepared standards or spiked samples. No human experts are described as establishing this analytical ground truth in the document.
For clinical performance (Expected Values): The "non AMI individuals" represent the "ground truth" for a normal population. The method for determining "non AMI" status is not detailed, but it would typically be established by physicians using clinical criteria (symptoms, EKG, other biomarkers). No specific number or qualification of experts is mentioned for this.

4. Adjudication method for the test set

Not applicable. This is an in vitro diagnostic device for quantitative determination of Myoglobin. The "test set" in this context refers to samples used to validate the device's analytical performance against known concentrations or a comparator device, not typically requiring human adjudication in the way an imaging AI algorithm might.

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

Not applicable. This is an in vitro diagnostic device, not an AI algorithm assisting human readers (e.g., radiologists) in interpreting medical images or clinical data. Therefore, an MRMC study is not relevant.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the study describes the standalone performance of the Immuno 1 Myoglobin method, which is an automated in vitro diagnostic assay. Its performance characteristics (imprecision, linearity, sensitivity, correlation with a comparator) are measured directly from the device's output. The "human-in-the-loop" equivalent would be the laboratory technician running the assay, but the output itself is quantitative and objective, not subject to human interpretation like imaging.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

Analytical Ground Truth:
- Known concentrations: For imprecision, interference, linearity, sample dilution, hook effect, recovery, and minimum detectable concentration, the ground truth was established by preparing samples with known, controlled concentrations of Myoglobin and/or interfering substances.
- Comparator Device: For correlation, the "ground truth" was derived from measurements obtained by a cleared predicate device, the Behring Nephelometer A.
Clinical Ground Truth:
- Clinical Status: For "Expected Values," the ground truth was the clinical status of "non AMI individuals," presumably diagnosed by standard clinical criteria, although details of this diagnosis are not provided.

8. The sample size for the training set

This document describes the performance of an in vitro diagnostic assay. Assays like the Immuno 1 Myoglobin method use calibrators to establish a dose-response curve, but this curve fitting is not analogous to "training" an AI algorithm with a large dataset. The six calibrators with specified Myoglobin concentrations (0, 60, 180, 600, 1500, and 3000 ng/mL) are used to generate the dose response curve. These calibrators are essentially the "training data" for the internal calculation process of the Immuno 1 system. The document does not specify a separate, distinct "training set" in the context of machine learning.

9. How the ground truth for the training set was established

As noted above, for this type of IVD, the "training set" refers to the calibrators. The ground truth for these calibrators (i.e., their Myoglobin concentrations) would be established through a rigorous manufacturing process, typically by gravimetric or volumetric methods, and validated against reference materials or established analytical techniques. The document provides the specific Myoglobin concentrations for each of the six calibrators, indicating these are known and controlled values.

Summary

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AUG 1 5 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

Assay Description

Image /page/0/Figure/10 description: The image shows a schematic representation of a technique with 5 steps. Step 1 shows 10 microliters of Immuno-Magnetic-Particles and 3 microliters of sample being pipetted into one cuvette. Step 2 shows 30 seconds later, 65 microliters of fluoresceinated capture and 65 microliters of alkaline phosphatase labeled marker antibody are added to the cuvette. Step 3 shows 15 minutes of incubation, step 4 shows washing four times, and step 5 shows adding substrate and starting readout, with the final result after 23 minutes.

Image /page/0/Figure/11 description: This image contains text describing a figure. The text states that the response read is directly proportional to the schematic representation of the Technicon Immuno 1 Myoglobin Assay. The figure is labeled as Fig. 1.

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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.

Image /page/1/Figure/3 description: The image shows a calibration curve for Myoglobin using Technicon Immuno 1. The graph plots concentration in ng/mL on the y-axis against AU/min on the x-axis, displaying a linear relationship. Data pairs are provided, showing values such as 0 ng/mL corresponding to 0.0019 AU/min and 3000 ng/mL corresponding to 3.1391 AU/min.

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

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 AssayData was collected on two Systems over twenty days with four replicates on each day on eachsystem
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 × x + 1.05

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

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

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Image /page/3/Figure/0 description: This figure is a scatter plot comparing BNA and Immuno 1, with values ranging from 0 to 4'000 on both axes. The data points are clustered along a diagonal, indicating a positive correlation between the two variables. There are also three trend lines plotted on the graph.

ﻣﺴﺘﻘ

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

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TEST.XLG (METHOD.XLD)

Method Comparison acc. to Bablok-Passing

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

Serum and Plasma Samples

Image /page/4/Figure/7 description: The image shows a scatter plot of 'Immuno 1' versus 'BNA', with data points, a regression line, and an identity line. The regression line has a slope of 1.04110 and an intercept of -0.98356. The number of samples is 54, and the sample type is 'all sample codes'. The confidence of correlation is 0.96814.

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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 interferantconcentration. 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:

)

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

- Triglycerides

Triglyceride Supertrate:	-2 Pool:	0 g/ dL
	+2 Pool:	- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------

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:

ﻠﻤﺴﺎ

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

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

sample content%	Identification	AU/min	conc.(meas.)	conc.(calc)	recovery (%)
100	Serum	1.1004	880.5	880.5	100
75	Sample A	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	2.7817	2420.1	2420.1	100
75	Sample B	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	3.1881	2887.9	2887.9	100
75	Sample C	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	2.7268	2360.9	2360.9	100
75	Sample D	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	2.6725	2302.7	2302.7	100
75	Sample E	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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Samplecontent %	Identification	AU/min	conc. (meas.)	conc. (calc)	dev.
100	Serum	1.0525	859.7	859.7	100
75	Sample A	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	2.7167	2344.3	2344.3	100
75	Sample B	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	3.0948	2765.1	2765.1	100
75	Sample C	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	2.8607	2499.2	2499.2	100
75	Sample D	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	2.7182	2345.6	2345.6	100
75	Sample E	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	1.44	1224.1	1224.1	100
75	Sample AA	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	PlasmaSample BB	2.6022	2332.7	2332.7	100
75	Sample BB	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	PlasmaSample CC	2.5736	2302.1	2302.1	100
75	Sample CC	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	PlasmaSample DD	1.2854	1090.7	1090.7	100
75	Sample DD	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	PlasmaSample EE	1.62	1382.2	1382.2	100
75	Sample EE	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		0.0032	-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	1.4613	1216.1	1216.1	100
75	Sample AA	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	-

. D

)

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Sample content	Identification	AU/min	conc (meas)	conc (calc)	dev (%)
%
100	Plasma	2.724	2392.9	2392.9	100
75	Sample BB	2.1735	1843.6	1794.675	97.3
50		1.4542	1210.1	1196.45	98.9
25		0.7596	639.2	598.225	93.5
10		0.283	242.9	239.29	98.5
0		0.0023	-0.2	0
100	Plasma	2.7637	2435.5	2435.5	100
75	Sample CC	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	1.3432	1117.6	1117.6	100
75	Sample DD	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	1.6146	1345.7	1345.7	100
75	Sample EE	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

ﻟﺮ

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Hook Effect

ﻤﺴﺎ

ﻣﺴﺎ

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.

Image /page/13/Figure/2 description: The image shows a graph with "ng/mL Myoglobin (expected)" on the x-axis and "ng/mL Myoglobin (measured)" on the y-axis. The graph shows a range labeled "Assay Range" between 100 and 10000 on the x-axis and 100 and 10000 on the y-axis. There is also a range labeled "Raw Data out of Range" between 10000 and 100000 on the x-axis. The graph shows a line with data points that increases from left to right, then decreases from left to right.

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	39.1	39.1	100.0
Sample α	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	38.5	38.5	100.0
Sample β	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	24.8	24.8	100.0
Sample γ	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	31.3	31.3	100.0
Sample δ	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 of the values were 88 ng/mL or less.

Immuno 1 Myoglobin: Normal Distribution

Image /page/15/Figure/4 description: This image is a graph titled "Fig. 5 Technicon Immuno 1 Myoglobin Assay: Normal". The graph shows concentration in ng/mL on the x-axis, ranging from 0 to 156. The y-axis shows a scale from 0 to 10. The graph shows a series of vertical bars, with the highest bars between 12 and 24 ng/mL.

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..

Regulation Number and Section

§ 866.5680 Myoglobin immunological test system.

(a)
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.(b)
Classification. Class II (performance standards).