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201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 1
Clinical ChemistrySpecific ProteinsPresentation
November 2004
AEROSET® and c8000® are registered trademarks of Abbott Laboratories.
All other trademarks, brands, trade names and product names are the property of their respective companies.
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 2
Agenda
Basic InformationSample HandlingReagent HandlingCalibrationQuality ControlReaction Methodology
Interfering SubstancesPrecisionMethod ComparisonAssay Specific Information Troubleshooting TipsQuestions & Answers
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 4
Specific Protein Assay HighlightsApolipoprotein A1
– Lipid transport – found in HDL and chylomicrons
Apolipoprotein B– Lipid transport - found in Non-HDL
lipoproteinsComplement C3
– Increased with acute phase reactions, AMI, cancer, pregnancy, viral hepatitis & diabetes
Complement C4– Increased in some malignancies
and acute phase reactionsHaptoglobin
– Binds Hemoglobin
Immunoglobulin A– Serum and body secretion
ImmunoglobulinImmunoglobulin G
– Secondary immune response– Found in blood, crosses placenta
Immunoglobulin M– Primary immune response
Prealbumin– T3, T4, and Vitamin A transport– Indicator of protein malnutrition
Transferrin– Iron Transport
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 5
Clinical Implications
Assay Decreased Levels Increased Levels Apolipoprotein A1 Atherosclerosis Low risk of coronary disease Apolipoprotein B Severe hepatic dysfunction Atherosclerosis, hyperlipidemias Complement (C3) AutoImmune disease, chronic
hepatitis, lupus Inflammatory Disease
Complement (C4) Autoimmune disease, chronic hepatitis, acute glomerular nephritis
Acute inflammatory process
Haptoglobin Hemolytic anemia, sickle cell anemia, liver disease
Acute and chronic inflammatory disease
Immunoglobulin (IgA) Immune deficiency states, non-IgA myelomas
IgA myelomea, chronic cirrhosis, chronic liver disease
Immunoglobulin (IgG) Immune deficiency states, non-IgG Myelomas
IgG myeloma, chronic infections, Liver disease
Immunoglobulin (IgM) Immune deficiency states, non-IgM myelomas
Waldenstrom’s macroglobulinemia, chronic infections, liver disease
Prealbumin Malnutrition, liver disease, acute Inflammation
Hodgkin’s disease, corticosteroid therapy
Transferrin Inflammation, chronic hepatitis Iron deficiency, acute hepatitis, pregnancy
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 7
Reagents
279R1: 3 x 20 mLR2: 3 x 8 mL
9D97-20Complement C4
Tests per KitKit configuration
List NumberReagent
230R1: 3 x 17 mLR2: 3 x 7 mL
9D91-20Haptoglobin
279R1: 3 x 20 mLR2: 3 x 8 mL
9D96-20Complement C3
243R1: 3 x 21 mLR2: 3 x 9 mL
9D93-20Apolipoprotein B
243R1: 3 x 21 mLR2: 3 x 9 mL
9D92-20ApolipoproteinA1
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 8
Reagents
373R1: 4 x 20 mLR2: 4 x 8 mL
9D98-20Immunoglobulin A
242R1: 3 x 18 mLR2: 3 x 6 mL
1E02-20Prealbumin
Tests per KitKit configuration
List NumberReagent
391R1: 5 x 20 mLR2: 5 x 9 mL
1E04-20Transferrin
373R1: 4 x 20 mLR2: 4 x 8 mL
1E01-20Immunoglobulin M
388R1: 4 x 20 mLR2: 4 x 20 mL
9D99-20Immunoglobulin G
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 9
Calibrators
1E78-02Specific Protein Multiconstituent
6E57-02Prealbumin
6E54-02ApoA1/ApoB
List NumberCalibrator
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 11
Sample Handling
Serum or Plasma* in glass or plastic tubes– *Prealbumin is serum only
Serum with or without gel barrierPlasma without a gel barrierAcceptable anticoagulants:
– Lithium Heparin– Ammonium Heparin– Sodium Heparin– EDTA
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Sample Handling
1 week2 weeks
2 to 8°C-20°C
Haptoglobin
3 days6 months
2 to 8°C-20°C
Immunoglobulin A (IgA)Immunoglobulin G (IgG)
PrealbuminTransferrin
5 days6 months
2 to 8°C-20°C
Immunoglobulin M (IgM)
2 days2 to 8°CComplement C4
8 days8 days
2 to 8°C-20°C
Complement C3
3 days2 months
1 year*
2 to 8°C-20°C-70°C
Apolipoprotein B
3 days2 months
2 to 8°C-20°C
Apolipoprotein A1Maximum StorageTemperatureAssay
1 * Thaw overnight at 2-8 °C
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Reagent Handling
Reagent is a liquid, ready-to-use, two-reagent kit.
The unopened reagents are stable until the expiration date whenstored at 2-8°C.Do not mix fresh reagent with in use reagent.Do not mix materials from different kit lot numbersMix gently, do not shake or foaming may occur. Due to small
reagent volumes, do not use a transfer pipette to remove bubbles.
Reagent onboard Stability
−IgA: 28 days
−IgG: 23 days
−ApoA, ApoB, C3, C4, Haptoglobin, IgM, Prealbumin, Transferrin: 57 days
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 15
Reagent Handling
R1 and R2 – both 20 mL bottlesIf running all 10 specific protein assays:Requires twenty 20 mL reagent cartridge adapters List Number 09D22-11, 10 adapters Same list number for AEROSET® and c8000®
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Reagent handling – c8000®
•R2 reagent carousel requires one large cartridge segment, B, C, D(L/N 04J31-01) to hold 20 mL round bottle reagent adapters.
•Two large cartridge segments are required to hold all 10 Specific Protein R2 reagents at the sametime.
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 17
c8000® Base configuration
R1R2
A
B
CD
BB
A
B
C
D
Segment D14 small positions
Segment C14 small positions
Segment B9 large positions
Segment A14 Small positions
R2
Segment D20 large positions
Segment C12 large positions
Segment B12 large positions
Segment A12 large positions
R1
BB
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 19
Calibration
IgA now uses 1:25 autodilution of highest calibrator (SP5) to create the lowest non-zero calibrator (C1)
Must edit linear high with highest calibrator value for all specific protein assays upon initial use and with each calibrator lot change.
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 20
CalibrationCalibration Stability
– ApoB: 38 days– IgA: 25 days– IgG: 23 days– ApoA1, C3, C4, Haptoglobin, IgM, Prealbumin,
Transferrin: 57 Days
Calibration Method– ApoA, ApoB, Prealbumin: Linear– C3, C4, Haptoglobin, IgA, IgG, IgM, Transferrin: Spline
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 21
Calibration– Preparation of Apo A1/Apo B calibrators
• Reconstitute with 1.0 mL Type II water• Allow to stand for 10 minutes• Dissolve by swirling – don’t shake• Prepare the four calibrator dilutions using the
reconstituted calibrator and diluent. – Reconstituted calibrator is stable for 2 weeks when
stored capped at 2 to 8°C– Diluted calibrators are stable for 1 day when stored
capped at 2 to 8°C
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 22
Apolipoprotein A1 Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 23
Apolipoprotein B Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 24
Complement C3 Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 25
Complement C4 Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 26
Haptoglobin Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 27
Immunoglobulin A Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 28
Immunoglobulin G Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 29
Immunoglobulin M Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 30
Prealbumin Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 31
Transferrin Calibration
AEROSET®
Conventional Units
c8000®
SI Units
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 33
Quality Control
Abbott control product is not available, however current AEROSET® peer group information is available with BioRad Immunology Plus. BioRad peer value information can be found on WWCS Webpage
– www.Bio-rad.com– US: 1-800-2-BIO-RAD– ROW: contact your local BioRad
Representative
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 34
Quality Control
IgA, IgG, IgM
IgA, IgG, IgM
ApoA, ApoB, C3, C4, Hapt,IgA, IgG, IgM, PAlb, TRF
Assays
360 (Trilevel, 12 x 5 mL)361, 362, 363 (12 x 5 mL)
LiquichekImmunoassay Plus
370 (Trilevel, 12 x 5 mL)371, 372, 373 (12 x 5 mL)
Lyphochek Immunoassay Plus
591, 592, 593 (6 x 1 mL)594, 595, 596 (6 x 3 mL)
Liquichek Immunoassay
Catalog #BioRad Control Name
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 36
Basic Principles in Turbidimetry and Nephelometry
When a soluble antigen is mixed in suitable proportions with the corresponding antibody a precipitate is formed. This reaction was described in quantitative terms in the pioneering work by Heidelberger & Kendall in 1929. In a now classical experiment, they showed that when increasing amounts of an antigen are added to a number of test tubes containing a constant quantity of the corresponding antibody, and the amount of precipitate was analyzed, a precipitin curve as shown was obtained.
Ag ConcentrationHeidelberger Curve
Ab Excess Zone Ag Excess Zone
Equivalence Zone
Imm
unop
reci
pita
te
Y
Y
Y
Y
Y
Y Y
Y
Y Y
Y Y
YYY
Quantitative Immunoprecipitin Curve
Y
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 37
Basic Principles in Turbidimetry and Nephelometry
Three zones:
1. The Antibody Excess Zone
The amount of precipitate increases as more antigen is added.
The supernatant still contains free antibody.
2. The Equivalence Zone
Maximum precipitation occurs.
The supernatant contains neither free antigen nor free antibody at the peak of the curve.
3. The Antigen Excess Zone
Due to high antigen concentration, the formation of small soluble immune complexes is favored rather than real precipitate. When insufficient amounts of antibody are available to bind with antigen, an erroneously low result can occur.
The supernatant contains free antigen.
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 38
Reaction MethodologyNephelometry (IN)Measures the intensity of scattered light (that which is reflected at various angles).
Turbidimetry (IT)Measures the intensity of light transmission (incident light beam passing through the cuvette).
Turbidimetry and Nephelometry Both are based on optical detection systems that measure the concentration of very small particles suspended in a liquid.Dilute antigen solutions are mixed with a solution of corresponding antibody to form immune complexes.Immune complexes cause solution turbidity.
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 39
Nephelometry or Turbidimetry?
"These two instrument approaches basically differ only in the nature of the optics. The basic principle is to measure that portion of an incident light beam which passes through thecuvette - Turbidimetry. Light beam reflected at various angles isNephelometry.
Both approaches have advanced greatly in the last decade and the instrument and computer controlled packages associated with the optical systems have produced devices which perform extremely well. "It is not possible to say which is better."
Each manufacturer has sought to deliver an instrument which is better than its competition, but in reality the comparisons are informative only when the kits themselves are examined."1
1Ritchie, Robert MD. Serum Proteins in Clinical Medicine, Vol. 1, Foundation of Blood Research. Scarborough, Maine. 1996. p. 2.0-4
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 40
Assay Reaction Sequence
Methodology – Immunoturbidimetric
Equation:
Analyte + PEG/buffer (R1) + Antibody to analyte (R2)(serum orplasma)
Turbidity due to analyte: antibody complex
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Reaction CheckProzone:Antigen excess. Extremely high immunoglobulin samples result in formation of soluble complexes instead of an insolublecomplex leading to incorrect results within the reportable range of the assay.
Reaction check: used with IgA and IgM assays to evaluate the reaction for prozone
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Prozone (Antigen Excess)Prozone (Antigen Excess)
– New IgA and IgM parameters will use a 1:5 sample dilution and the RCD flag as the standard configuration to reduce the likelihood of prozone. Auto Rerun/Retest is used to rerun LL or < samples undiluted.
IgM High Sample Neat vs 1:5 Absorbance Data
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
read point
abso
rban
ce Sample 1 neathi callo calsample 1 1:5
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 44
Prozone (Antigen Excess)Precision of 1:5 dilution IgA and IgM
% CV Goal = 4.1 % CV Goal = 4.4
Cntl Total CV
Cntl Total CV
1 0.95 1 1.4
2 1.1 2 1.31
IgA Neat
3 1.43
IgM Neat
3 1.32
1 0.98 1 1.16
2 0.62 2 1.04
c8000®
IgA Diluted
3 0.8
c8000®
IgM Diluted
3 1.44
1 1.34 1 1.99
2 1.35 2 1.71
AEROSET®
IgA Diluted
3 1.27
AEROSET®
IgM Diluted
3 1.43
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 45
IgA and IgG sensitivity improvements
– IgA sensitivity was improved by using a 1:25 auto dilution of Cal 5 to create a new low calibrator between zero and Cal 1
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 46
IgA and IgG sensitivity improvements
IgG sensitivity was improved by auto retest of samples < 320 mg/dL with 3X sample volume.
IgG % Recovery vs Target
0255075
100125150175200
0 100 200 300 400 500
IgG mg/dL
Obs
erve
d %
of T
arge
t
2.7 uL Svol8.1 uL Svol
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 47
Percent Extrapolation (%EXT Flag)(AEROSET ® only)
%EXT flagging is based on the absorbance value (or absorbance change) of the highest calibrator. The absorbance value (or absorbance change) is defined as the difference between the absorbance (or absorbance change) of the highest calibrator and the reagent blank (absorbance or absorbance change).
Determines the numerical value beyond which results will be flagged if they exceed that percentage of the absorbance or rate of the highest calibrator.
The value for this parameter is 1% for all specific protein assays.
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 48
Percent Extrapolation (%EXT Flag)(AEROSET ® only)
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Apolipoprotein A1 Interfering SubstancesInterfering
Substances Concentration Target
Conc % of Target
Bilirubin 15 mg/dL 143.4 93.5
30 mg/dL 143.4 85.6
Hemoglobin 1000 mg/dL 152.3 92.0
2000 mg/dL 152.3 84.3
Human Trig 500 mg/dL 165.2 90.6
750 mg/dL 165.2 86.0
Intralipid 1000 mg/dL 150.5 100.7
2000 mg/dL 150.5 99.9
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 51
Apolipoprotein B - Interfering Substances
Interfering Substances
Concentration Target Conc
% of Target
Bilirubin 30 mg/dL 97.7 98.8
60 mg/dL 97.7 96.7
Hemoglobin 1000 mg/dL 90.9 101.2
2000 mg/dL 90.9 102.0
Human Trig 750 mg/dL 102.0 102.8
1000 mg/dL 102.0 104.6
Intralipid 1000 mg/dL 102.1 101.7
2000 mg/dL 102.1 101.9
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 52
Complement C3- Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2
Interfering Substances
Concentration Target Conc
% of Target Target Conc % of Target
Bilirubin 30 mg/dL 72.4 97.6 174.7 99.9
60 mg/dL 72.4 96.1 174.7 97.8
Hemoglobin 1000 mg/dL 60.4 94.0 187.1 98.5
2000 mg/dL 60.4 95.9 187.1 99.4
Human Trig 750 mg/dL 75.7 104.8 182.7 99.6
1000 mg/dL 75.7 102.2 182.7 98.4
Intralipid 1000 mg/dL 63.4 98.0 170.8 100.2
2000 mg/dL 63.4 89.0 170.8 97.8
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 53
Complement C4- Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2
Interfering Substances
Concentration Target Conc
% of Target Target Conc % of Target
Bilirubin 30 mg/dL 13.8 97.5 50.5 95.7
60 mg/dL 13.8 96.7 50.5 94.5
Hemoglobin 1000 mg/dL 13.6 90.6 35.8 96.7
2000 mg/dL 13.6 92.2 35.8 104.5
Human Trig 750 mg/dL 14.2 104.0 50.2 99.5
1000 mg/dL 14.2 101.5 50.2 101.0
Intralipid 1000 mg/dL 12.4 96.8 44.6 100.1
2000 mg/dL 12.4 90.8 44.6 97.0
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 54
Haptoglobin - Interfering SubstancesInterfering
Substances Concentration Target
Conc % of Target
Bilirubin 30 mg/dL 171.3 96.6
60 mg/dL 171.3 93.2
Hemoglobin 1000 mg/dL 138.9 90.1
2000 mg/dL 138.9 89.8
Human Trig 750 mg/dL 175.7 103.2
1000 mg/dL 175.7 102.6
Intralipid 1000 mg/dL 133.1 99.8
2000 mg/dL 133.1 97.1
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 55
IgA - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2
Interfering Substances
Concentration Target Conc
% of Target Target Conc % of Target
Bilirubin 30 mg/dL 81.9 99.5 496.3 101.9
60 mg/dL 81.9 98.4 496.3 98.8
Hemoglobin 1000 mg/dL 81.5 98.1 514.7 98.4
2000 mg/dL 81.5 96.3 514.7 96.0
Human Trig 750 mg/dL 85.1 98.6 516.4 89.7
1000 mg/dL 85.1 97.4 516.4 85.4
Intralipid 1000 mg/dL 83.1 96.9 459.5 99.5
2000 mg/dL 83.1 69.9 459.5 88.2
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 56
IgG - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2
Interfering Substances
Concentration Target Conc
% of Target Target Conc % of Target
Bilirubin 30 mg/dL 695.7 98.18 1934.1 98.62
60 mg/dL 695.7 96.32 1934.1 98.19
Hemoglobin 1000 mg/dL 582.9 98.70 1624.2 101.00
2000 mg/dL 582.9 96.64 1624.2 101.41
Human Trig 750 mg/dL 875.8 99.99 2025.2 95.90
1000 mg/dL 875.8 99.35 2025.2 92.97
Intralipid 1000 mg/dL 668.0 100.7 1849.2 100.7
2000 mg/dL 668.0 101.3 1849.2 100.5
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 57
IgM - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2
Interfering Substances
Concentration Target Conc
% of Target
Concentration Target Conc
% of Target
Bilirubin 30 mg/dL 50.8 109.79 30 mg/dL 270.6 99.64
60 mg/dL 50.8 108.00 60 mg/dL 270.6 98.33
Hemoglobin 250 mg/dL 53.9 92.28 1000 mg/dL 223.9 96.89
500 mg/dL 53.9 88.20 2000 mg/dL 223.9 95.20
Human Trig 750 mg/dL 62.2 100.33 750 mg/dL 290.5 101.81
1000 mg/dL 62.2 100.44 1000 mg/dL 290.5 101.83
Intralipid 1000 mg/dL 47.7 97.80 1000 mg/dL 275.6 99.65
2000 mg/dL 47.7 85.37 2000 mg/dL 275.6 95.14
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 58
Prealbumin - Interfering SubstancesInterfering
Substances Concentration Target
Conc % of Target
Bilirubin 30 mg/dL 14.7 96.4
60 mg/dL 14.7 92.3
Hemoglobin 500 mg/dL 13.6 91.2
750 mg/dL 13.6 88.6
Human Trig 750 mg/dL 18.6 97.4
1000 mg/dL 18.6 98.6
Intralipid 1000 mg/dL 13.0 95.7
2000 mg/dL 13.0 64.0
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 59
Transferrin - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2
Interfering Substances
Concentration Target Conc
% of Target Target Conc
% of Target
Bilirubin 30 mg/dL 256.3 98.1 341.7 100.3
60 mg/dL 256.3 94.3 341.7 98.1
Hemoglobin 1000 mg/dL 213.3 102.7 278.6 100.3
2000 mg/dL 213.3 101.4 278.6 101.2
Human Trig 750 mg/dL 275.7 101.7 373.6 98.7
1000 mg/dL 275.7 101.1 373.6 97.3
Intralipid 1000 mg/dL 231.1 100.3 294.6 99.4
2000 mg/dL 231.1 99.4 294.6 101.4
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Apolipoprotein A1- Precision (< 4.9 % Total CV)
Level N Mean mg/dL
Within Run SD
Within Run %CV
Total SD
Total %CV
Level 1 80 66.2 1.27 1.9 2.11 3.2 Level 2 80 188.3 2.17 1.2 5.10 2.7 Level 3 80 208.8 2.46 1.2 3.85 1.8
Level N Mean mg/dL
Within Run SD
Within Run %CV
Total SD
Total %CV
Level 1 80 43.4 1.93 4.4 2.75 6.3 Level 2 80 98.3 1.26 1.3 1.72 1.8 Level 3 80 140.3 1.89 1.3 5.53 3.9
Apolipoprotein B - Precision (< 6.5 % Total CV)
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 62
Complement C3 - Precision (< 3.9 % Total CV)Level N Mean
mg/dLWithin
Run SDWithin Run
%CV Total SD
Total %CV
Level 1 80 68.3 1.53 2.2 2.11 3.1 Level 2 80 138.5 1.73 1.3 1.85 1.3 Level 3 80 201.7 2.51 1.2 6.50 3.2
Level N Mean mg/dL
Within Run SD
Within Run %CV
Total SD
Total %CV
Level 1 80 13.4 0.33 2.5 0.49 3.7 Level 2 80 25.9 0.29 1.1 0.38 1.5 Level 3 80 37.4 0.73 2.0 1.34 3.6
Complement C4 - Precision (< 4.5 % Total CV)
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 63
Haptoglobin - Precision (< 6.0 % Total CV)
Level N Mean mg/dL
Within Run SD
Within Run %CV
Total SD
Total %CV
Level 1 80 59.4 0.75 1.3 3.35 5.6 Level 2 80 122.7 1.87 1.5 2.60 2.1 Level 3 80 182.3 1.79 1.0 4.49 2.5
Immunoglobulin A - Precision (< 4.1 % Total CV)Level N Mean
mg/dLWithin
Run SDWithin Run
%CV Total SD
Total %CV
Level 1 80 104.4 1.22 1.2 1.40 1.3 Level 2 80 206.9 2.15 1.0 2.79 1.4 Level 3 80 307.7 2.56 0.8 3.91 1.3
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 64
Immunoglobulin G - Precision (< 3.4 % Total CV)Level N Mean
mg/dLWithin
Run SDWithin Run
%CV Total SD
Total %CV
Level 1 80 948.3 10.08 1.1 20.14 2.1 Level 2 80 1639 22.64 1.4 30.84 1.9 Level 3 80 2993.1 81.93 2.7 90.88 3.0
Immunoglobulin M - Precision (< 4.4 % Total CV)Level N Mean
mg/dLWithin
Run SDWithin Run
%CV Total SD
Total %CV
Level 1 80 50.1 1.24 2.5 1.35 2.7 Level 2 80 135.9 2.19 1.6 2.33 1.7 Level 3 80 187.3 3.41 1.8 3.86 2.1
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 65
Prealbumin - Precision (< 5.5 % Total CV)Level N Mean
mg/dLWithin
Run SDWithin Run
%CV Total SD
Total %CV
Level 1 80 13.4 0.12 0.9 0.38 2.9 Level 2 80 21.2 0.18 0.9 0.28 1.3 Level 3 80 31.2 0.80 2.6 1.07 3.4
Transferrin - Precision (< 5.0 % Total CV)Level N Mean
mg/dLWithin
Run SDWithin Run
%CV Total SD
Total %CV
Level 1 80 143.7 2.46 1.7 6.58 4.6 Level 2 80 220.2 2.51 1.1 3.72 1.7 Level 3 80 325.7 5.22 1.6 10.83 3.3
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 67
Method Comparison
20.2 to 269.343.3 to 190.35.8 to 335.226.9 to 196.8Range (mg/dL)
-6.33.1-4.83.3Mean % Bias
1.01.060.990.98Slope
0.9960.9790.9960.992Corr Coef
-5.56-2.92-5.596.24Y-Intercept
93809580N
c8000® vs. AEROSET®
AEROSET®
vs. Hitachic8000® vs. AEROSET®
AEROSET®
vs. Hitachi
Apolipoprotein BApolipoprotein A1
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 68
Method Comparison
4.7 to 59.510.8 to 59.012.0 to 357.127.8 to 235.6Range (mg/dL)
-3.50.2-2.78.3Mean % Bias
0.991.000.941.03Slope
0.9990.9960.9990.994Corr Coef
-0.470.023.977.73Y-Intercept
89679980N
c8000® vs. AEROSET®
AEROSET®
vs. Hitachic8000® vs. AEROSET®
AEROSET®
vs. Hitachi
Complement C4Complement C3
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 69
Method Comparison
18.3 to 3351.417.0 to 518.613.4 to 279.01.8 to 276.8Range (mg/dL)
2.51.8-4.15.0Mean % Bias
0.991.050.981.03Slope
1.0000.9980.9990.998Corr Coef
7.41-5.45-0.880.25Y-Intercept
101879147N
c8000® vs. AEROSET®
AEROSET®
vs. Hitachic8000® vs. AEROSET®
AEROSET®
vs. Hitachi
Immunoglobulin AHaptoglobin
201020-101 Clinical Chemistry Specific Proteins Presentation, version 1 70
Method Comparison
14.2 to 1698.810.2 to 244.4123.8 to 3856.2
401.6 to 2943.6
Range (mg/dL)
-6.89.2-1.5-1.2Mean % Bias
0.961.040.990.96Slope
1.0000.9980.9990.997Corr Coef
-2.185.24-8.7640.56Y-Intercept
93789773N
c8000® vs. AEROSET®
AEROSET®
vs. Hitachic8000® vs. AEROSET®
AEROSET®
vs. Hitachi
Immunoglobulin MImmunoglobulin G
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Method Comparison
21.7 to 541.732.1 to 418.93.2 to 56.24.3 to 44.1Range (mg/dL)
-3.53.0-0.65.7Mean % Bias
0.971.041.011.05Slope
0.9880.9950.9980.996Corr Coef
-0.38-1.48-0.450.09Y-Intercept
114809580N
c8000® vs. AEROSET®
AEROSET®
vs. Hitachic8000® vs. AEROSET®
AEROSET®
vs. Hitachi
TransferrinPrealbumin
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Assay Specific Information
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Apolipoprotein A1Assay Method:
ImmunoturbidimetricReagent List #: 9D92-20Calibraton:
Method: LinearApo A1/Apo B Calibrator: 6E54-02
Assay Stabilityonboard Stability: 57 Days(1368 hours)Calibration Stability: 57 Days (1368 Hours)
Assay VolumesSpecimen: 2 uLR1 Reagent: 200 uLR2 Reagent: 67 uL
Reportable range: 16 mg/dL (0.16 g/L) up to highest calibrator
Limit of Quantitation: < 3 mg/dL (0.03 g/L)
Acceptable specimen: plasma or serum
Anticoagulants Tested:Lithium heparin, ammonium heparin, sodium heparin, EDTA
Precision: < 4.9 % Total CVNew Information
1:2 dilution protocol addedCalibration interval extended to 57 daysLH field contains lowest possible value for high calibrator
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Apolipoprotein BAssay Method: ImmunoturbidimetricReagent List#: 9D93-20Calibraton:
Method: LinearApo A1/Apo B Calibrator: 6E54-02
Assay StabilityOnboard Stability: 57 Days (1368 hours)Calibration Stability: 38 Days(912 Hours)
Assay VolumesSpecimen: 2 uLR1 Reagent: 200 uLR2 Reagent: 67 uL
Reportable range: 11 mg/dL (0.11 g/L) up to highest calibrator
Limit of Quantitation: < 3 mg/dL (0.03 g/L)
Acceptable specimen: plasma or serum
Anticoagulants Tested:Lithium heparin, ammonium heparin, sodium heparin, EDTA
Precision: < 6.5 % Total CVNew Information
1:4 Dil 1 protocol addedLH field contains lowest possible value for high calibrator
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Complement C3
Assay Method: ImmunoturbidimetricReagent List#: 9D96-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay StabilityOnboard Stability: 57 Days(1368 hours)Calibration Stability: 57 Days (1368 Hours)
Assay VolumesSpecimen: 3 uLR1 Reagent: 167 uLR2 Reagent: 47 uL
Reportable range: 11 mg/dL (0.11 g/L) up to highest calibrator
Limit of Quantitation: < 5 mg/dL (0.05 g/L)
Acceptable specimen: plasma or serum
Anticoagulants Tested:Lithium heparin, ammonium heparin, sodium heparin, EDTA
Precision: < 3.9 % Total CVNew Information
Reference ranges adjusted to reflect recent literatureCalibration interval extended to 57 daysLH field contains lowest possible value for high calibrator
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Complement C4Assay Method: ImmunoturbidimetricReagent List#: 9D97-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay Stabilityonboard Stability: 57 Days (1368 hours)Calibration Stability: 57 Days(1368 Hours)
Assay VolumesSpecimen: 3 uLR1 Reagent: 167 uLR2 Reagent: 47 uL
Reportable range: 2.9 mg/dL (0.029 g/L) up to highest calibrator
Limit of Quantitation: < 1.0 mg/dL (0.01 g/L)
Precision: < 4.5 % Total CVAcceptable specimen: plasma or
serumAnticoagulants Tested:
Lithium heparin, ammonium heparin, sodium heparin, EDTA
New InformationReference ranges adjusted to reflect recent literatureLH field contains lowest possible value for high calibratorCalibration interval extended to 57 days
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HaptoglobinAssay Method:
ImmunoturbidimetricReagent List#: 9D91-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay Stabilityonboard Stability: 57 Days(1368 hours)Calibration Stability: 57 Days(1368 Hours)
Assay VolumesSpecimen: 2 uLR1 Reagent: 167 uLR2 Reagent: 47 uL
Reportable range: 8 mg/dL (0.08 g/L) up to highest calibrator
Limit of Quantitation: < 4 mg/dL (0.04 g/L)
Precision: < 6.0 % Total CVAcceptable specimen: plasma or
serumAnticoagulants Tested:
Lithium heparin, ammonium heparin, sodium heparin, EDTA
New Information1:4 Dil 2 protocol addedCal interval extended to 57 daysLH field contains lowest possible value for high calibrator
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Immunoglobulin A (IgA)Reportable range:
5 mg/dL (0.05 g/L) up to highest calibrator
Limit of Quantitation: < 3 mg/dL (0.03 g/L)Precision: < 4.1 % Total CVAcceptable specimen: plasma or serumAnticoagulants Tested:
Lithium heparin, ammonium heparin, sodium heparin, EDTA
New InformationNo longer need to run in panel with IgA_D and IgA_R Samples are run at a 1:5 dilution initially (sample volume has increased from 6.5 uL to 20 uL)Added rerun rule for LL to rerun with Dil 1Reaction check parameters added to detectprozoneHigh calibrator is auto-diluted for C1LH field contains lowest possible value for high calibrator LL field changed from 31 to 5 mg/dLAdded 1:10 Dil 2 parameters
Assay Method: ImmunoturbidimetricReagent List#: 9D98-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay Stabilityonboard Stability: 28 Days (672 hours)Calibration Stability: 25 Days(600 Hours)
Assay VolumesSpecimen: 20 uLStandard dilution: 1:5R1 Reagent: 167 uLR2 Reagent: 47 uL
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Immunoglobulin G (IgG)Reportable range:
109 mg/dL (1.09 g/L) up to highest calibrator
Limit of Quantitation: < 61 mg/dL (0.61 g/L)Precision: < 3.4 % Total CVAcceptable specimen: plasma or serumAnticoagulants Tested:
Lithium heparin, ammonium heparin, sodium heparin, EDTA
New InformationLL changed from 194 mg/dL (1.94 g/L) to 320 mg/dL (3.20 g/L). Dil 1 protocol added to use 3x the standard sample volume extending the lower limit of reportable range to 109 mg/dL (1.09 g/L)Rerun rule added to use Dil 1 for samples < 320 mg/dLReference ranges adjusted to reflect recent literature
Assay Method: ImmunoturbidimetricReagent List#: 9D99-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay Stabilityonboard Stability: 23 Days (552 hours)Calibration Stability: 23 Days (552 hours)
Assay VolumesSpecimen: 2.7 uLR1 Reagent: 160 uLR2 Reagent: 160 uL
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Immunoglobulin M (IgM)Reportable range:
5 mg/dL (0.05 g/L) up to highest calibrator
Limit of Quantitation: < 2 mg/dL (0.02 g/L)
Precision: < 4.4 % Total CVAcceptable specimen: plasma or serumAnticoagulants Tested:
Lithium heparin, ammonium heparin, sodium heparin, EDTA
New InformationReaction check range changedLinear low changed from 14 mg/dL (0.14 g/L) to 5 mg/dL (.05 g/L)Samples are run at a 1:5 dilution initially (sample volume has increased from 3.0 uL to 20 uL)Dil 1 protocol runs samples undilutedAdded rerun rule to use Dil 1 for LL samples Calibration interval extended to 57 days
Assay Method: ImmunoturbidimetricReagent List#: 1E01-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay Stabilityonboard Stability: 57 Days (1368 hours)Calibration Stability: 57 Days (1368 Hours)
Assay VolumesSpecimen: 20 uLStandard dilution: 1:5R1 Reagent: 167 uLR2 Reagent: 47 uL
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Prealbumin
Assay Method: ImmunoturbidimetricReagent List#: 1E02-20Calibraton:
Method: LinearPrealbumin Calibrator: 6E57-02
Assay Stabilityonboard Stability: 57 Days (1368 hours)Calibration Stability: 57 Days (1368 Hours)
Assay VolumesSpecimen: 3 uLR1 Reagent: 167 uLR2 Reagent: 33 uL
Reportable range: 3 mg/dL (0.03 g/L) up to highest calibrator
Limit of Quantitation: < 1 mg/dL (0.01 g/L)
Precision: < 5.5 % Total CVAcceptable specimen: serum
onlyNew Information
Calibration interval extended to 57 days
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TransferrinAssay Method: ImmunoturbidimetricReagent List#: 1E04-20Calibraton:
Method: SplineSpecific Protein MCC: 1E78-02
Assay Stabilityonboard Stability: 57 Days(1368 hours)Calibration Stability: 57 Days(1368 Hours)
Assay VolumesSpecimen: 2 uLR1 Reagent: 200 uLR2 Reagent: 67 uL
Reportable range: 19 mg/dL (0.19 g/L) up to highest calibrator
Limit of Quantitation: < 9 mg/dL (0.09 g/L)
Precision: < 5.0 % Total CVAcceptable specimen: plasma or
serumAnticoagulants Tested:
Lithium heparin, ammonium heparin, sodium heparin, EDTA
New InformationReference ranges adjusted to reflect recent literatureCalibration interval extended to 57 days
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Troubleshooting Tips
IgG Imprecision– R1 Reagent: 160 uL, R2 Reagent: 160 uL,
large volume in cuvettes– Check:
• Mixers• R2 sample or reagent probe, syringes,
wash, mixer, tubing
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Troubleshooting Tips
IgM or Prealbumin Imprecision– Any assay using 340nm as the primary
wavelength is more sensitive to lamp age.– IgM and PAlb are the only specific protein assays
using this wavelength (340/700)– View the reaction graph, primary wavelength
only.– Normal lamp function: Flat line between reads 2
and 16 and a smooth line bending from vertical to horizontal between reads 17 and 33
– Lamp degradation: Progress curve not smooth (exhibits an erratic saw-tooth appearance)
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Troubleshooting Tips
All Specific Protein assays– Active ingredient is in R2 component of 2
reagent assay systems– R2 dispense and mixing drives the
reaction– For precision concerns, verify R2 mixer
alignment and function, R2 reagent probe dispense and position, and R2 reagent syringe seal tips and o-ring.
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Troubleshooting Tips
IgA and IgM– Saline (0.85% or 0.90% sodium chloride)
is used as the diluent for the standard dilution
– When diluent is not onboard:• AEROSET® – assay button will have
black text indicating illegal assay and it will not run until diluent is loaded.
• c8000® – Error code 0218, Unable to process test, no Processing Modules available
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Troubleshooting TipsBarcode errors
• Round bottles may rotate slightly in adapter when the reagent carousel rotates, moving the bar code label and affecting the ability of the bar code scanner to read the bar code label.
– Turn the bottle to allow the bar coded scanner to read the reagent bar code label
– Insert the bottle from the bottom of the adapter so the label is not scraped.
• Bottles may fit tightly in some adapters. Frequent installation and removal of bottles may damage barcode.
• If the adaptor is not seated correctly the bottle may tilt resulting in barcode errors.
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Questions and AnswersWhy are linearity claims only up to the highest calibrator?
– For non-linear assays (C3, C4, Hapt, IgA, IgG, IgM and TRF) accuracy by recovery cannot be guaranteed beyond the highest calibrator. AEROSET® uses %EXT to prevent extrapolation and c8000® does not print results above the highest calibrator for non-linear assays.
– For linear assays (ApoA, ApoB and PAlb) accuracy above the highest calibrator is dependant on calibrator concentrations which vary from lot to lot. AEROSET® uses %EXT and c8000®
uses the LH parameter to prevent extrapolation. Even though R&D testing supports linearity beyond the high calibrator, linearity was defined as up to the high calibrator. Data from alarge cohort of samples indicate very few samples will exceed the high calibrator concentration.
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Questions and Answers
Why are the calibration modes for ApoA, ApoB, and PAlb linear if the calibration curves are not straight?
Although the graphs appear non-linear for these assays, the calibrations approximate a linear response closely enough to insure accurate results across the calibration range. Therefore the calibration modes have been defined as linear for each of these assays.
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Questions and AnswersWhen the AEROSET® launched the Specific Protein assays, only two levels of controls were run for precision. Now, why are three levels of controls shown for precision?
– Commercial 3 level controls were used for the c8000® precision testing. The level 3 control used for c8000® testing is approximately the same concentration as the level 2 control used in the original AEROSET® testing.
– The revised package inserts now contain a combination of AEROSET® and/or c8000® data. The data was selected based on the highest Total %CV for each control level independent of the instrument it was tested on.
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Questions and Answers
When a manual dilution is performed for the IgA and IgM assays, what value do I enter for the sample dilution?
– Enter the manual dilution factor only. The analyzer will automatically adjust for the use of a standard sample dilution.
Example, a manual 1:4 dilution is prepared and tested using the standard sample dilution (1:5). Enter 4 for the manual dilution and the analyzer will automatically multiply the measured concentration by 20 (manual dilution X standard sample dilution) to determine the result.
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Questions and Answers
Why are the linear low and LOQ not the same?– LOQ is the lowest analyte concentration at
which the CV=20%. – Some Specific Protein assays become
increasingly inaccurate as analyte concentrations drop below the lowest calibrator and approach LOQ. For this reason the linear low limit for these assays is based on accuracy by recovery studies rather than LOQ studies.
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Questions and Answers
When a LH (AEROSET®) or > (c8000®) error code is observed when running the IgA or IgM assay, what should the customer do next?
The sample needs to be diluted and rerun. An autodilution can be performed using Dil 2 (AEROSET®) or 1:10 (c8000®). c8000® Operations Manual does not direct to dilute and rerun. P5-218
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Questions and Answers
When a LL (AEROSET®) or < (c8000®) error code is observed when running the IgA or IgM assay, what should the customer do next?
The sample needs to be rerun undiluted. An autodilution can be performed using Dil 1 (AEROSET®) or Undiluted (c8000®). c8000®
Operations Manual does not direct to dilute and rerun. P5-218
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Questions and Answers
Why would a LH (AEROSET®) or > (c8000®) flag be observed when a result is less than the highest calibrator?
Operator failed to edit the LH field to equal the high calibrator concentration for a new lot of calibrators. Both the calibrator concentrations and the linear high field must be edited as specified in the calibrator value sheet.
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Questions and Answers
• Why are the following c8000® results observed for the IgM assay? (highest calibrator is 330 mg/dL(3.30 g/L)
Std (1:5) = >330 mg/dL. Repeat Manual 1:5 dilution =1856 mg/dL
Spline assays, when run using the Standard (1:5) sample dilution on the c8000®, will not correct the result for the dilution factor. The analyzer should have multiplied the LH field by the dilution factor and printed the result for the Std (1:5) as > 1650 mg/dL. This will be corrected in a future version of the software.