P reliminary Communication S pecial Focus I ssue: M atrix
effects in
LBA s
For reprint orders, please contact [email protected]
An ultra-sensitive clinical biomarker assay: quantitation of thymus and activation-regulated chemokine in human plasma Background: Thymus and activation-regulated chemokine (TARC) is a Th2 type, pro-allergic secreted chemokine. TARC in plasma/serum has been proposed as a marker for disease activity of atopic dermatitis (AD) and as a pharmacodynamic readout in the clinical development of novel agents for the treatment of AD. Results: An ultra-sensitive electrochemiluminescence assay for TARC in human plasma was developed and analytically validated. The assay demonstrated excellent performance characteristics, including precision, sensitivity, dilution linearity, accuracy and specificity. Stability and biological variability of TARC in plasma were also assessed for clinical sample analysis and data interpretation. Conclusion: The improved sensitivity allowed the measurement of approximately 90% TARC inhibition from baseline levels of healthy subjects and >90% TARC inhibition from baseline levels of AD patients after drug treatment. A validated TARC electrochemiluminescence assay enables pharmacodynamic assessment in the development of AD therapeutics. Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease associated with elevated serum IgE levels and eosinophilia [1]. The unique feature of inflammation is the infiltration of specific leukocyte subsets from the blood into affected tissue [2]. Acute lesional skin in AD is histologically characterized by the predominant infiltration of Th2-type CD4+ memory-effector T cells [3]. This process is orchestrated by chemo kines and their receptors. Chemokines are small (8000–10,000 Da) secreted proteins that are important in immune responses and inflammatory reactions as they selectively recruit subsets of leukocytes that express specific chemokine receptors. Two members of the CC chemokine subfamily, thymus and activation-regulated chemokine (TARC/CCL17) and macrophagederived chemokine (MDC/CCL22), are highaffinity ligands for CC chemokine receptor 4 (CCR4, CD194), which is expressed on different subsets of T cells: Th2 cells, cutaneous leukocyte-associated antigen skin-homing T cells, and CD25+ T-suppressor cells [4]. TARC and MDC serve the recruitment and migration of CCR4expressing cells. These two ligands for CCR4 are not just redundant; instead, they appear to have specific roles. In inflamed skin lesions, TARC initially acts on CCR4 at the endothelial surface to facilitate vascular recognition, whereas MDC subsequently interacts with the receptor within the tissue microenvironment to direct cellular localization [5].
The Th2-type chemokine TARC is constitutively and abundantly expressed in the thymus [6], and is produced by monocytederived dendritic cells [7–9], endothelial cells [10] and epidermal keratinotyes [11]. In humans, elevated levels of TARC have been observed in serum or plasma of patients with AD compared with healthy control subjects [12–16]. In addition, TARC levels in serum or plasma of patients with AD correlated significantly with the objective SCORAD score, a scoring index to assess the severity of AD [17,18], indicating plasma/serum TARC as a disease activity marker for AD. Furthermore, TARC levels in serum or plasma of patients with AD decreased after treatment in accordance with the improvement of clinical symptoms [12–14,19], suggesting that plasma/serum TARC can also be utilized as a pharmacodynamic (PD) biomarker to assess disease pathway engagement in the clinical development of AD therapeutics. In this article, we describe the optimization and fit-for-purpose validation of an ultra-sensitive electrochemiluminescence (ECL) assay for TARC quantitation in human plasma, including the assessment of assay dilution linearity, precision, accuracy, sensitivity, specificity, stability and biological variability. This validated assay allows us to measure TARC as a PD marker in early-stage clinical studies, involving healthy subjects and disease patients, of novel agents to treat AD.
10.4155/BIO.14.72 © 2014 Future Science Ltd
Bioanalysis (2014) 6(8), 1069–1080
Xuemei Zhao*, Liliana Delgado, Russell Weiner & Omar F Laterza Molecular Biomarkers & Diagnostics Laboratory, Merck Research Laboratories, RY50A-300, 126 East Lincoln Avenue, Rahway, NJ 07065, USA *Author for correspondence: Tel.:+1 732 594 1747 [email protected]
ISSN 1757-6180
1069
P reliminary Communication | Key Term Pharmacodynamic biomarker: Biomarker of a
certain pharmacological response, which are of special interest in dose optimization studies in early drug development.
Zhao, Delgado, Weiner & Laterza
Experimental Reagents Recombinant human TARC was purchased from R&D Systems, Inc. (MN, USA; catalog number: 364-DN). Human chemokine 9-plex kit was purchased from Meso Scale Discovery® (MSD; MD, USA; catalog number: K15001C-2). The kit was supplied with human chemokine 9-plex plate (catalog number: N05001B-1), SULFOTAG detection antibody blend (catalog number: D2002–3), human chemokine 9-plex calibrator blend (ultra-sensitive; catalog number: C3001–2), Diluent 2 (catalog number: R51BB-4), Diluent 3 (catalog number: R51BA-4) and Read Buffer T (4X) (catalog number: R92TC-3). Human TARC ultra-sensitive kit was purchased from MSD (catalog number: K151BGC-2). The kit was supplied with cytokine panel 2 ultra-sensitive plate (catalog number: N45016A-1), human TARC detection antibody (catalog number: D21BG-3), cytokine panel 2 calibrator blend (catalog number: C0016– 2), Diluent 2 (catalog number: R51BB-3), Diluent 3 (catalog number: R51BA-5), and MSD Read Buffer T (4X; catalog number: R92TC-3). Dulbecco’s phosphate-buffered saline (PBS), 1X was purchased from Mediatech, Inc. (VA, USA; catalog number: 21–031-CV). Surfact-Amps 20 (10% Tween 20) was purchased from Thermo Scientific (IL, USA; product number: CAS 9005–64–5). Blocker Casein in PBS (1% [w/v] casein) was purchased from Thermo Scientific (product number: 37528). Heterophilic Blocking Reagent 1 (HBR1; Scantibodies Laboratory, Inc.; CA, USA; part number: 3KC534–075). StabilZyme SELECT® Stabilizer was purchased from SurModics (MN, USA; product number: SZ03–1000). Distilled water was purchased from Life Technologies (NY, USA; catalog number: 15230–147). EDTA plasma samples of healthy control subjects were purchased from Bioreclamation, Inc. (NY, USA). EDTA plasma samples of healthy control subjects from blood draw on five different days from each subject were purchased from BioChemed Services (VA, USA). EDTA plasma samples of patients with AD were purchased from ProteoGenex, Inc. (CA, USA). EDTA plasma samples of healthy control subjects, as well as patients with mild, moderate, severe and very severe AD were purchased from PrecisionMed, Inc. (CA, USA). Sample
collection Human plasma and serum samples from apparently healthy subjects were obtained with 1070
Bioanalysis (2014) 6(8)
informed consent. For plasma preparation, whole blood was drawn into purple/lavender top vacutainer tubes. The blood tubes were mixed by gentle inversion ten times, incubated at room temperature (RT) for up to 2 h, and subsequently centrifuged at 1500 × g for 15 min at RT. EDTA plasma samples were collected, aliquoted and stored at -80°C. For serum preparation, whole blood was drawn into red top vacutainer tubes. The blood tubes were incubated at RT for 30 min and then centrifuged at 1500 × g for 15 min at RT. Serum samples were collected, aliquoted and stored at -80°C. Freeze–thaw
treatment of plasma samples A freeze–thaw treatment of plasma samples was executed in the following procedure. Samples were stored at -80°C for at least 4 h. They were then removed from the -80°C freezer and thawed at RT for 2 h. After a complete freeze–thaw cycle, samples were either analyzed in the TARC assay or stored at -80°C. Preparation
of standards & QC samples Escherichia coli-derived recombinant human TARC protein (Ala24-Ser94) was used as standard for the TARC assay. The recombinant protein lacks the first 23 amino acid of human TARC. Recombinant human TARC protein (1 vial, 25 µg) was reconstituted in 2.5 ml of StabilZyme SELECT® Stabilizer to a final concentration of 10 µg/ml. Then 200 µl of 10 µg/ml TARC was mixed with 199.8 ml of Casein-T (1% Casein and 0.05% Tween 20 in PBS) to obtain the TARC standard stock at 10,000 pg/ml. The TARC standard stock was aliquoted and stored at -80°C. Prior to each ana lysis, standard samples were prepared by serial (threefold) dilution of the TARC standard stock with Casein-T resulting in a nine-point standard curve with a range of 1.5 to 10,000 pg/ml. Four sets of QC samples (extra high, high, medium and low) were prepared by spiking recombinant human TARC protein into a human plasma sample purchased from Bioreclamation, Inc. The QC samples were aliquoted and stored at -80°C. TARC
ECL assay procedure & sample analysis All samples (plasma, serum, standards, QC and blank) were analyzed in duplicate on a MSDsupplied 96-well plate following the same procedure. Assay buffer was used as blank. All reagents were brought to RT prior to analysis. future science group
Quantitation of thymus & activation-regulated chemokine in plasma
| Preliminary Communication
Table 1. LLOD (pg/ml) of thymus and activation-regulated chemokine and macrophage-derived chemokine in human plasma. Buffer
Thymus and activationregulated chemokine
Macrophage-derived chemokine
Meso Scale Discovery® Diluent 2 3% bovine serum albumin in phosphate-buffered saline 0.05% Tween 20 Heterophilic blocking reagent 1 Casein 0.05% Tween 20 Heterophilic blocking reagent 1
50 13
415 6.5
9
255
Table 2. Intra- and inter-assay precision in plasma QC samples. Intra-assay† n Mean (pg/ml) SD %CV
Inter-assay‡
Extra high QC
High QC
Medium QC
Low QC
Extra high QC High QC
Medium QC
Low QC
10 1916.2 63.5 3.3
9 407.3 22.8 5.6
10 194.8 4.4 2.3
10 46.6 1.8 3.9
19 1896.4 69.8 3.7
19 203.9 8.5 4.2
19 44.9 2.4 5.3
19 382.7 15.1 4.0
n = Nine or ten samples. Each sample was run in duplicate. n = 19 runs.
† ‡
Plasma/serum samples and QC samples were diluted 1:4 in a (Casein-T + HBR) buffer containing 0.4 mg/ml HBR1 in Casein-T (1.2 µg of HBR1 per µl of plasma or serum). A plate with 150 µl Casein per well was incubated for 1.5 h with vigorous shaking (1000 × g) at RT. The plate was then washed three times with 200 µl per well of PBS-T (0.05% Tween 20 in PBS). After the addition of 50 µl Casein-T per well and 50 µl of standard, diluted sample or QC sample, the plate was incubated at RT for 2 h with vigorous shaking (1000 × g). Upon completion of the sample incubation, the plate was washed with PBS-T buffer three times, and 25 µl of human TARC detection antibody was added to each well. The plate was incubated for 2 h with vigorous shaking (1000 rpm) at RT. The plate was again washed with PBS-T buffer three times. After the addition of 150 µl of 2X Read Buffer T (1:2 dilution of 4X Read Buffer T in distilled water) to each well, the plate was read on the MSD Sector® Imager 6000.
Software
& statistical analysis The MSD Discovery Workbench® analysis software was used for data acquisition and data ana lysis. The standard curve was modeled using the least squares fitting algorithm, which allowed back-calculation of the TARC concentration in the samples from standards with known levels of TARC. In addition, signals from standards with known levels of TARC were used to calculate the concentration of TARC in the sample. The software utilized a four-parameter logistic model and included a 1/Y2 weighting function to determine the mean, standard deviation (SD) and %CV. Back-calculated concentrations and percentage difference of the back-calculated concentrations were calculated using Microsoft Excel. Results & discussion An optimized ECL assay Initially, we evaluated the MSD human chemokine 9-plex kit with the goal to simultaneously measure TARC and MDC, two Th2 chemokines involved
Table 3. Sensitivity: determination of LLOQ. Sample
LLOQ_2, 1:2 dilution
LLOQ_1
LLOQ_2
LLOQ_3
LLOQ_4
n Mean (pg/ml) SD %CV
8 11.3 1.4 12.6
12 14.3 1.3 8.9
12 24.2 1.9 7.8
6 43.7 2.2 5.0
6 61.9 3.3 5.4
future science group
www.future-science.com
1071
P reliminary Communication |
Zhao, Delgado, Weiner & Laterza
Table 4. Dilution linearity of human plasma in sample diluent. Sample dilution Thymus and activation% Difference from regulated chemokine† (pg/ml) neat plasma Extra high QC Neat 2 4 8 16 32
1774.5 1808.7 1803.7 1926.2 1951.4 1937.1
0.0 1.9 1.6 8.5 10.0 9.2
131.0 151.9 159.3 159.8 177.5
effects in
LBA s
For reprint orders, please contact [email protected]
An ultra-sensitive clinical biomarker assay: quantitation of thymus and activation-regulated chemokine in human plasma Background: Thymus and activation-regulated chemokine (TARC) is a Th2 type, pro-allergic secreted chemokine. TARC in plasma/serum has been proposed as a marker for disease activity of atopic dermatitis (AD) and as a pharmacodynamic readout in the clinical development of novel agents for the treatment of AD. Results: An ultra-sensitive electrochemiluminescence assay for TARC in human plasma was developed and analytically validated. The assay demonstrated excellent performance characteristics, including precision, sensitivity, dilution linearity, accuracy and specificity. Stability and biological variability of TARC in plasma were also assessed for clinical sample analysis and data interpretation. Conclusion: The improved sensitivity allowed the measurement of approximately 90% TARC inhibition from baseline levels of healthy subjects and >90% TARC inhibition from baseline levels of AD patients after drug treatment. A validated TARC electrochemiluminescence assay enables pharmacodynamic assessment in the development of AD therapeutics. Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease associated with elevated serum IgE levels and eosinophilia [1]. The unique feature of inflammation is the infiltration of specific leukocyte subsets from the blood into affected tissue [2]. Acute lesional skin in AD is histologically characterized by the predominant infiltration of Th2-type CD4+ memory-effector T cells [3]. This process is orchestrated by chemo kines and their receptors. Chemokines are small (8000–10,000 Da) secreted proteins that are important in immune responses and inflammatory reactions as they selectively recruit subsets of leukocytes that express specific chemokine receptors. Two members of the CC chemokine subfamily, thymus and activation-regulated chemokine (TARC/CCL17) and macrophagederived chemokine (MDC/CCL22), are highaffinity ligands for CC chemokine receptor 4 (CCR4, CD194), which is expressed on different subsets of T cells: Th2 cells, cutaneous leukocyte-associated antigen skin-homing T cells, and CD25+ T-suppressor cells [4]. TARC and MDC serve the recruitment and migration of CCR4expressing cells. These two ligands for CCR4 are not just redundant; instead, they appear to have specific roles. In inflamed skin lesions, TARC initially acts on CCR4 at the endothelial surface to facilitate vascular recognition, whereas MDC subsequently interacts with the receptor within the tissue microenvironment to direct cellular localization [5].
The Th2-type chemokine TARC is constitutively and abundantly expressed in the thymus [6], and is produced by monocytederived dendritic cells [7–9], endothelial cells [10] and epidermal keratinotyes [11]. In humans, elevated levels of TARC have been observed in serum or plasma of patients with AD compared with healthy control subjects [12–16]. In addition, TARC levels in serum or plasma of patients with AD correlated significantly with the objective SCORAD score, a scoring index to assess the severity of AD [17,18], indicating plasma/serum TARC as a disease activity marker for AD. Furthermore, TARC levels in serum or plasma of patients with AD decreased after treatment in accordance with the improvement of clinical symptoms [12–14,19], suggesting that plasma/serum TARC can also be utilized as a pharmacodynamic (PD) biomarker to assess disease pathway engagement in the clinical development of AD therapeutics. In this article, we describe the optimization and fit-for-purpose validation of an ultra-sensitive electrochemiluminescence (ECL) assay for TARC quantitation in human plasma, including the assessment of assay dilution linearity, precision, accuracy, sensitivity, specificity, stability and biological variability. This validated assay allows us to measure TARC as a PD marker in early-stage clinical studies, involving healthy subjects and disease patients, of novel agents to treat AD.
10.4155/BIO.14.72 © 2014 Future Science Ltd
Bioanalysis (2014) 6(8), 1069–1080
Xuemei Zhao*, Liliana Delgado, Russell Weiner & Omar F Laterza Molecular Biomarkers & Diagnostics Laboratory, Merck Research Laboratories, RY50A-300, 126 East Lincoln Avenue, Rahway, NJ 07065, USA *Author for correspondence: Tel.:+1 732 594 1747 [email protected]
ISSN 1757-6180
1069
P reliminary Communication | Key Term Pharmacodynamic biomarker: Biomarker of a
certain pharmacological response, which are of special interest in dose optimization studies in early drug development.
Zhao, Delgado, Weiner & Laterza
Experimental Reagents Recombinant human TARC was purchased from R&D Systems, Inc. (MN, USA; catalog number: 364-DN). Human chemokine 9-plex kit was purchased from Meso Scale Discovery® (MSD; MD, USA; catalog number: K15001C-2). The kit was supplied with human chemokine 9-plex plate (catalog number: N05001B-1), SULFOTAG detection antibody blend (catalog number: D2002–3), human chemokine 9-plex calibrator blend (ultra-sensitive; catalog number: C3001–2), Diluent 2 (catalog number: R51BB-4), Diluent 3 (catalog number: R51BA-4) and Read Buffer T (4X) (catalog number: R92TC-3). Human TARC ultra-sensitive kit was purchased from MSD (catalog number: K151BGC-2). The kit was supplied with cytokine panel 2 ultra-sensitive plate (catalog number: N45016A-1), human TARC detection antibody (catalog number: D21BG-3), cytokine panel 2 calibrator blend (catalog number: C0016– 2), Diluent 2 (catalog number: R51BB-3), Diluent 3 (catalog number: R51BA-5), and MSD Read Buffer T (4X; catalog number: R92TC-3). Dulbecco’s phosphate-buffered saline (PBS), 1X was purchased from Mediatech, Inc. (VA, USA; catalog number: 21–031-CV). Surfact-Amps 20 (10% Tween 20) was purchased from Thermo Scientific (IL, USA; product number: CAS 9005–64–5). Blocker Casein in PBS (1% [w/v] casein) was purchased from Thermo Scientific (product number: 37528). Heterophilic Blocking Reagent 1 (HBR1; Scantibodies Laboratory, Inc.; CA, USA; part number: 3KC534–075). StabilZyme SELECT® Stabilizer was purchased from SurModics (MN, USA; product number: SZ03–1000). Distilled water was purchased from Life Technologies (NY, USA; catalog number: 15230–147). EDTA plasma samples of healthy control subjects were purchased from Bioreclamation, Inc. (NY, USA). EDTA plasma samples of healthy control subjects from blood draw on five different days from each subject were purchased from BioChemed Services (VA, USA). EDTA plasma samples of patients with AD were purchased from ProteoGenex, Inc. (CA, USA). EDTA plasma samples of healthy control subjects, as well as patients with mild, moderate, severe and very severe AD were purchased from PrecisionMed, Inc. (CA, USA). Sample
collection Human plasma and serum samples from apparently healthy subjects were obtained with 1070
Bioanalysis (2014) 6(8)
informed consent. For plasma preparation, whole blood was drawn into purple/lavender top vacutainer tubes. The blood tubes were mixed by gentle inversion ten times, incubated at room temperature (RT) for up to 2 h, and subsequently centrifuged at 1500 × g for 15 min at RT. EDTA plasma samples were collected, aliquoted and stored at -80°C. For serum preparation, whole blood was drawn into red top vacutainer tubes. The blood tubes were incubated at RT for 30 min and then centrifuged at 1500 × g for 15 min at RT. Serum samples were collected, aliquoted and stored at -80°C. Freeze–thaw
treatment of plasma samples A freeze–thaw treatment of plasma samples was executed in the following procedure. Samples were stored at -80°C for at least 4 h. They were then removed from the -80°C freezer and thawed at RT for 2 h. After a complete freeze–thaw cycle, samples were either analyzed in the TARC assay or stored at -80°C. Preparation
of standards & QC samples Escherichia coli-derived recombinant human TARC protein (Ala24-Ser94) was used as standard for the TARC assay. The recombinant protein lacks the first 23 amino acid of human TARC. Recombinant human TARC protein (1 vial, 25 µg) was reconstituted in 2.5 ml of StabilZyme SELECT® Stabilizer to a final concentration of 10 µg/ml. Then 200 µl of 10 µg/ml TARC was mixed with 199.8 ml of Casein-T (1% Casein and 0.05% Tween 20 in PBS) to obtain the TARC standard stock at 10,000 pg/ml. The TARC standard stock was aliquoted and stored at -80°C. Prior to each ana lysis, standard samples were prepared by serial (threefold) dilution of the TARC standard stock with Casein-T resulting in a nine-point standard curve with a range of 1.5 to 10,000 pg/ml. Four sets of QC samples (extra high, high, medium and low) were prepared by spiking recombinant human TARC protein into a human plasma sample purchased from Bioreclamation, Inc. The QC samples were aliquoted and stored at -80°C. TARC
ECL assay procedure & sample analysis All samples (plasma, serum, standards, QC and blank) were analyzed in duplicate on a MSDsupplied 96-well plate following the same procedure. Assay buffer was used as blank. All reagents were brought to RT prior to analysis. future science group
Quantitation of thymus & activation-regulated chemokine in plasma
| Preliminary Communication
Table 1. LLOD (pg/ml) of thymus and activation-regulated chemokine and macrophage-derived chemokine in human plasma. Buffer
Thymus and activationregulated chemokine
Macrophage-derived chemokine
Meso Scale Discovery® Diluent 2 3% bovine serum albumin in phosphate-buffered saline 0.05% Tween 20 Heterophilic blocking reagent 1 Casein 0.05% Tween 20 Heterophilic blocking reagent 1
50 13
415 6.5
9
255
Table 2. Intra- and inter-assay precision in plasma QC samples. Intra-assay† n Mean (pg/ml) SD %CV
Inter-assay‡
Extra high QC
High QC
Medium QC
Low QC
Extra high QC High QC
Medium QC
Low QC
10 1916.2 63.5 3.3
9 407.3 22.8 5.6
10 194.8 4.4 2.3
10 46.6 1.8 3.9
19 1896.4 69.8 3.7
19 203.9 8.5 4.2
19 44.9 2.4 5.3
19 382.7 15.1 4.0
n = Nine or ten samples. Each sample was run in duplicate. n = 19 runs.
† ‡
Plasma/serum samples and QC samples were diluted 1:4 in a (Casein-T + HBR) buffer containing 0.4 mg/ml HBR1 in Casein-T (1.2 µg of HBR1 per µl of plasma or serum). A plate with 150 µl Casein per well was incubated for 1.5 h with vigorous shaking (1000 × g) at RT. The plate was then washed three times with 200 µl per well of PBS-T (0.05% Tween 20 in PBS). After the addition of 50 µl Casein-T per well and 50 µl of standard, diluted sample or QC sample, the plate was incubated at RT for 2 h with vigorous shaking (1000 × g). Upon completion of the sample incubation, the plate was washed with PBS-T buffer three times, and 25 µl of human TARC detection antibody was added to each well. The plate was incubated for 2 h with vigorous shaking (1000 rpm) at RT. The plate was again washed with PBS-T buffer three times. After the addition of 150 µl of 2X Read Buffer T (1:2 dilution of 4X Read Buffer T in distilled water) to each well, the plate was read on the MSD Sector® Imager 6000.
Software
& statistical analysis The MSD Discovery Workbench® analysis software was used for data acquisition and data ana lysis. The standard curve was modeled using the least squares fitting algorithm, which allowed back-calculation of the TARC concentration in the samples from standards with known levels of TARC. In addition, signals from standards with known levels of TARC were used to calculate the concentration of TARC in the sample. The software utilized a four-parameter logistic model and included a 1/Y2 weighting function to determine the mean, standard deviation (SD) and %CV. Back-calculated concentrations and percentage difference of the back-calculated concentrations were calculated using Microsoft Excel. Results & discussion An optimized ECL assay Initially, we evaluated the MSD human chemokine 9-plex kit with the goal to simultaneously measure TARC and MDC, two Th2 chemokines involved
Table 3. Sensitivity: determination of LLOQ. Sample
LLOQ_2, 1:2 dilution
LLOQ_1
LLOQ_2
LLOQ_3
LLOQ_4
n Mean (pg/ml) SD %CV
8 11.3 1.4 12.6
12 14.3 1.3 8.9
12 24.2 1.9 7.8
6 43.7 2.2 5.0
6 61.9 3.3 5.4
future science group
www.future-science.com
1071
P reliminary Communication |
Zhao, Delgado, Weiner & Laterza
Table 4. Dilution linearity of human plasma in sample diluent. Sample dilution Thymus and activation% Difference from regulated chemokine† (pg/ml) neat plasma Extra high QC Neat 2 4 8 16 32
1774.5 1808.7 1803.7 1926.2 1951.4 1937.1
0.0 1.9 1.6 8.5 10.0 9.2
131.0 151.9 159.3 159.8 177.5
