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QUANTA Flash M2 (MIT3) is a chemiluminescent immunoassay for the semi-quantitative determination of IgG antimitochondrial antibodies in human serum. The presence of antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of primary biliary cholangitis.

QUANTA Flash M2 (MIT3) Calibrators are intended for use with the QUANTA Flash M2 (MIT3) chemiluminescent immunoassay for the determination of IgG anti-mitochondrial antibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.

QUANTA Flash M2 (MIT3) Controls are intended for use with the QUANTA Flash M2 (MIT3) chemiluminescent immunoassay for quality control in the determination of IgG anti-mitochondrial antibodies in human serum.

Device Description

The QUANTA Flash M2 (MIT3) assay is designed to run on the BIO-FLASH® instrument. This platform is a fully automated closed system with continuous load and random access capabilities that automatically processes the samples, runs the assay and reports the results. It includes liquid handling hardware, luminometer and computer with software-user interface. The QUANTA Flash M2 (MIT3) assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.

Recombinant MIT3 antigen is coated onto paramagnetic beads. The bead suspension is lyophilized and stored in the bead tube. Prior to use in the BIO-FLASH system, the sealed reagent tubes are pierced with the reagent cartridge lid and the beads are rehydrated and resuspension buffer by pipetting up and down with a transfer pipette. The reagent cartridge is then loaded onto the BIO-FLASH instrument. Samples are also loaded onto the instrument in samples are diluted by the BIO-FLASH with system rinse in a small disposable plastic cuvette. Small amounts of the diluted patient serum, the beads, and assay buffer are combined into a second cuvette, and mixed. This cuvette is then incubated at 37°C. The beads are magnetized and washed several times. Isoluminol conjugated anti-human IgG antibodies are then added to the cuvette, and again incubated at 37°C. The beads are magnetized and washed repeatedly. The isoluminol conjugate is oxidized when Trigger 1 (Fe(II)coproporphyrin in sodium hydroxide solution) and Trigger 2 (urea-hydrogen peroxide in sodium chloride solution) are added to the cuvette, and the flash of light produced from this reaction is measured as Relative Light Units (RLU) by the BIO-FLASH optical system. The RLU are proportional to the amount of isoluminol conjugate that is bound to the human IgG, which is in turn proportional to the amount of anti-M2 (MIT3) antibodies bound to the corresponding beads.

For quantitation, the QUANTA Flash M2 (MIT3) assay utilizes a predefined lot specific Master Curve that is uploaded onto the instrument through the reagent cartridge barcode. Every new lot number of reagent cartridge must be calibrated before first use, with the QUANTA Flash M2 (MIT3) Calibrators. Based on the results obtained with the three Calibrators included in the Calibrator Set (sold separately), an instrument specific Working Curve is created, which is used to calculate chemiluminescent units (CU) from the instrument signal (RLU) obtained for each sample.

The QUANTA Flash M2 (MIT3) kit contains the following materials:

One (1) QUANTA Flash M2 (MIT3) Reagent Cartridge One (1) vial of Resuspension buffer One (1) Transfer pipette

The QUANTA Flash M2 (MIT3) reagent cartridge contains the following reagents for 50 determinations:

M2 (MIT3) coated paramagnetic beads, lyophilized. a.
b. Assay buffer - colored pink, containing Tris-buffered saline, Tween 20, protein stabilizers and preservatives.
Tracer IgG Isoluminol labeled anti-human IgG antibodies in buffer, containing protein C. stabilizers and preservative.

The QUANTA Flash M2 (MIT3) Calibrators kit contains two vials each of Calibrator 2, and Calibrator 3:

QUANTA Flash M2 (MIT3) Calibrators:

QUANTA Flash M2 (MIT3) Calibrator 1: Two (2) barcode labeled tubes containing 0.3 mL prediluted, ready to use reagent. Calibrators contain human anti-mitochondrial antibodies in stabilizer and preservative.
-QUANTA Flash M2 (MIT3) Calibrator 2: Two (2) barcode labeled tubes containing 0.3 mL prediluted, ready to use reagent. Calibrators contain human anti-mitochondrial antibodies in stabilizer and preservative.
-QUANTA Flash M2 (MIT3) Calibrator 3: Two (2) barcode labeled tubes containing 0.3 mL prediluted, ready to use reagent. Calibrators contain human anti-mitochondrial antibodies in stabilizer and preservative.

The QUANTA Flash M2 (MIT3) Controls kit contains two vials of Negative Control and two vials of Positive Control:

QUANTA Flash M2 (MIT3) Controls:

QUANTA Flash M2 (MIT3) Negative Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human anti-mitochondrial antibodies in stabilizer and preservative.
QUANTA Flash M2 (MIT3) Positive Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human anti-mitochondrial antibodies in stabilizer and preservative.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study information for the QUANTA Flash M2 (MIT3) device:

1. Table of Acceptance Criteria and Reported Device Performance

Feature/Metric	Acceptance Criteria	Reported Device Performance
Precision	Total %CV: < 10%	Within Run: CV(%) for samples varied from 2.6% to 5.5%. Between Runs: CV(%) for samples varied from 1.1% to 3.6%. Between Days: CV(%) for samples varied from 0.0% to 3.9%. Total: CV(%) for samples varied from 2.9% to 7.6%. (All within <10%)
Reproducibility (Between Sites)	Total %CV: < 10%	CV(%) for 9 samples varied from 2.1% to 9.1%. (All within <10%)
Reproducibility (Between Lots)	All %CV values were within the acceptance limit, 10%.	CV(%) for 6 samples varied from 4.0% to 8.7%. (All within <10%)
Linearity	1. Best fitting polynomial is a linear one. 2. Otherwise, the difference between the best-fitting nonlinear and linear polynomial is less than 20%, or ± 4 CU, whichever is greater (allowable nonlinearity).	All six specimens fulfilled the acceptance criteria, showing good linearity in their respective ranges. Slope (95% CI): Ranged from 0.96 to 1.08. Y-Intercept (95% CI): Ranged from -146.8 to 26.0. R2: All 1.00. Average % Recovery: Ranged from 98.5% to 109.2%. (All within acceptable limits).
Interference	85% - 115% recovery, or ± 4 CU difference, whichever is greater.	No interference was detected with: - Bilirubin up to 1 mg/mL (recovery: 93% to 100%) - Hemoglobin up to 2 mg/mL (recovery: 94% to 101%) - Triglycerides up to 1000 mg/dL (recovery: 94% to 102%) - Cholesterol up to 332.5 mg/dL (recovery: 97% to 104%) - Human IgG up to 35 mg/mL (recovery: 91% to 115%, or 1.8 CU to 3.7 CU) - Ursodeoxycholic acid up to 0.75 mg/mL (recovery: 97% to 104%) - Prednisone up to 0.3 mg/mL (recovery: 100% to 105%) - Methotrexate up to 9.1 mg/mL (recovery: 96% to 103%) - Cholestyramine up to 18 mg/mL (recovery: 97% to 100%) - Colestipol up to 18 mg/mL (recovery: 98% to 104%) - Hydroxyzine up to 1 mg/mL (recovery 100% to 108%) - Glutamate Oxacatate Transaminase up to 0.12 IU/mL (recovery: 98% to 115%) - HDL Cholesterol up to 3.5 mg/mL (recovery: 98% to 105%) - Rheumatoid factor up to 153.4 IU/mL (recovery: 96% to 111%). (All within acceptable ranges).
Sample Stability	90-110% average recovery.	All samples fulfilled the acceptance criteria at each time point for each condition (up to 21 days at 2-8°C, up to 48 hours at room temperature, and up to 3 freeze/thaw cycles).
Reagent Shelf Life (Beads)	Lower 95% Cl interval of regression line is ≥85% and upper 95% Cl interval is ≤115% at day 14; no individual data point has ≤75% or ≥125% recovery at day 14.	All three lots of beads retained between 85% and 115% reactivity (considering the 95% Cl) after two weeks at 37 ± 3℃.
Reagent Shelf Life (Controls/Calibrators)	Lower 95% Cl interval of regression line is ≥90% and upper 95% Cl interval is ≤110% at day 14; no individual data point has ≤80% or ≥120% recovery at day 14.	All Calibrators and Controls maintained between 90% and 110% reactivity (considering the 95% Cl) when stored at 37 ± 3°C for 2 weeks.
In-use Stability (Calibrators)	1. All four calibrations performed in the 8 hour period are successful. 2. Mean Calibrator RLU recovery values for the first 4 calibrations are between 90% and 110% compared to the first use. 3. Control/patient panel CU recovery values are between 85% and 115% of those obtained on the first calibration curve.	The first four calibrations performed in the 8 hour period were considered valid. Calibrators yielded average RLU recovery values from 100% to 110%. Control/patient panel CU recovery ranged from 87.8% to 96.6%. (Supports claim of up to 4 calibrations over an 8-hour period).
In-use Stability (Controls)	1. All values run within their established range. 2. Linear regression line obtained by plotting %recovery values against the number of runs stays between 85% and 115% at run 15.	All controls ran within their respective acceptable ranges for all runs. The regression line remained between 85% and 115% at run 15 for both Controls. (Supports claim of up to 15 uses, at 10 minutes onboard per use).
In-use Stability (Reagent Cartridge)	Stability claim established at: - Actual measurement day preceding the 95% confidence interval of regression line reaches 85% or 115% recovery, OR - Actual measurement day preceding the day when 2 data points or ≥2% of recovery data (whichever is greater) is ≤ 75% or ≥ 125% recovery.	RP0006 lot: 70 days 151003 lot: 60 days. In-use (onboard) stability was set at 60 days.
Real Time Stability	Reagent Cartridge/Negative & Positive Controls: % recovery of each sample between 80-120% (compared to baseline). %CV of replicates <15%. Calibrators: % recovery of trilpicates average between 85% and 115%. %CV of triplicates <10%. Controls: results should fall within their acceptable ranges.	All results to date (up to 9 months for reagent cartridge and controls, 3 months for calibrators at time of submission) were within the acceptance limits.
Clinical Sensitivity	(Not explicitly stated as a separate acceptance criterion, but presented in the context of clinical performance evaluation against predicate.)	84.1% (77.4 - 89.1% CI) for Primary Biliary Cholangitis (PBC)
Clinical Specificity	(Not explicitly stated as a separate acceptance criterion, but presented in the context of clinical performance evaluation against predicate.)	99.0% (97.6 - 99.6% CI) for Controls (non-PBC patient groups)

2. Sample Size and Data Provenance for Test Set (Clinical Validation Study)

Sample Size: A total of 586 characterized samples were included in the Validation Set for the QUANTA Flash M2 (MIT3).
- For clinical sensitivity and specificity calculations, 568 samples were used (excluding 14 Limited cutaneous systemic sclerosis (ISSc) samples and 4 AIH/PBC overlap samples).
Data Provenance: Not explicitly stated regarding country of origin. The samples were "characterized" and included various patient groups (e.g., Autoimmune Hepatitis, Primary Sclerosing Cholangitis, Liver Cancer, Celiac Disease, Hepatitis B/C, Syphilis, Ulcerative Colitis, Crohn's Disease, Alcoholic liver disease, Idiopathic inflammatory myopathies, Systemic lupus erythematosus, Sjögren's syndrome, Sicca syndrome, Type 1 Diabetes, Osteoporosis, Chronic fatigue, Skin conditions, Drug-induced hepatotoxicity, Hypothyroidism, PBC). This suggests the data is retrospective, from existing sample banks of patients with known diagnoses.

3. Number of Experts and Qualifications for Ground Truth (Test Set)

The document states that the "cohort of characterized samples" were used for clinical validation. It also mentions "three diagnosed PBC patient specimens were assayed to aid in the determination of the cut-off."
However, the document does not explicitly state the number of experts used to establish the ground truth or their specific qualifications (e.g., radiologist with 10 years of experience) for the clinical validation set. The characterization implies that the samples had a pre-established clinical diagnosis, likely by medical professionals (e.g., physicians, pathologists), but the details of this process are not provided.

4. Adjudication Method (Test Set)

The document does not describe an adjudication method (e.g., 2+1, 3+1, none) for the clinical validation test set. The diagnoses for the "characterized samples" are presented as given.

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

No Multi-Reader Multi-Case (MRMC) comparative effectiveness study was done. This is an in vitro diagnostic device for semi-quantitative determination of antibodies, not an imaging device typically evaluated with MRMC studies involving human readers.

6. Standalone (Algorithm Only) Performance Study

Yes, a standalone study was done. The entire clinical validation and analytical performance sections evaluate the device (QUANTA Flash M2 (MIT3)), i.e., the algorithm/instrument system, without human interpretation beyond the initial sample collection and result reading from the instrument. The results presented for sensitivity, specificity, precision, linearity, etc., are all measures of the device's standalone performance.

7. Type of Ground Truth Used

The ground truth for the clinical validation study was based on clinical diagnoses of "characterized samples" from various patient groups, including confirmed Primary Biliary Cholangitis (PBC) patients and control groups for specificity. It also mentions "diagnosed PBC patient specimens" for cut-off determination. This falls under a combination of clinical diagnosis and presumed outcomes data (i.e., whether the patient actually had the disease).

8. Sample Size for Training Set

The document mentions a "reference population for establishing the reference interval for the M2 (MIT3) assay consisted of 180 subjects" for establishing the cut-off. This could be considered a "development set" or part of fine-tuning the algorithm's decision threshold (cut-off).
It also states "three diagnosed PBC patient specimens were assayed to aid in the determination of the cut-off."
Beyond this, the document does not explicitly describe a separate, distinct "training set" for the core algorithm in the way a machine learning model might. The analytical characterization and master curve development are part of the intrinsic design and calibration of the assay.

9. How Ground Truth for Training Set Was Established

For the reference interval/cut-off establishment:
- The 180 subjects included: 100 apparently healthy blood donors, 30 infectious disease patients, 30 rheumatoid arthritis patients, and 20 celiac disease patients.
- The cut-off was established in accordance with CLSI EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline - Third Edition.
- The Analyse-it for Excel software was used for calculations.
- The non-parametric percentile method was used due to non-normal distribution of results.
- Three diagnosed PBC patient specimens were also assayed to aid in determining the cut-off, ensuring optimal differentiation.
- The "Master Curve" for quantitation is generated during manufacturing using 7 "Master Curve Standards" with assigned CU values. These assigned values serve as the ground truth for calibrating the system's quantitative output.

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