clinical chemistry serum proteins - yeec · 201020-101 clinical chemistry specific proteins...

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.


Agenda

Basic InformationSample HandlingReagent HandlingCalibrationQuality ControlReaction Methodology

Interfering SubstancesPrecisionMethod ComparisonAssay Specific Information Troubleshooting TipsQuestions & Answers


Basic Information


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


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


Ordering Information


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


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


Calibrators

1E78-02Specific Protein Multiconstituent

6E57-02Prealbumin

6E54-02ApoA1/ApoB

List NumberCalibrator


Sample Handling


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


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


Reagent Handling


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


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®


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.


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


Calibration


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.


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


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


Apolipoprotein A1 Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Apolipoprotein B Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Complement C3 Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Complement C4 Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Haptoglobin Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Immunoglobulin A Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Immunoglobulin G Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Immunoglobulin M Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Prealbumin Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Transferrin Calibration

AEROSET®

Conventional Units

c8000®

SI Units


Quality Control


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

http://www.bio-rad.com/

http://www.bio-rad.com/


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


Reaction Methodology


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


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.


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.


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


Assay Reaction Sequence

Methodology – Immunoturbidimetric

Equation:

Analyte + PEG/buffer (R1) + Antibody to analyte (R2)(serum orplasma)

Turbidity due to analyte: antibody complex


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


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


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


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


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


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.


Percent Extrapolation (%EXT Flag)(AEROSET ® only)


Interfering Substances


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


Apolipoprotein B - Interfering Substances


Concentration Target Conc

% of Target


60 mg/dL 97.7 96.7


2000 mg/dL 90.9 102.0


1000 mg/dL 102.0 104.6


2000 mg/dL 102.1 101.9


Complement C3- Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2



% 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


Complement C4- Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2




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


Haptoglobin - Interfering SubstancesInterfering


Conc % of Target


60 mg/dL 171.3 93.2


2000 mg/dL 138.9 89.8


1000 mg/dL 175.7 102.6


2000 mg/dL 133.1 97.1


IgA - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2




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


IgG - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2




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


IgM - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2



% of Target


% 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


Prealbumin - Interfering SubstancesInterfering


Conc % of Target


60 mg/dL 14.7 92.3


750 mg/dL 13.6 88.6


1000 mg/dL 18.6 98.6


2000 mg/dL 13.0 64.0


Transferrin - Interfering SubstancesMedical Decision Level 1 Medical Decision Level 2



% 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


Precision


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


Apolipoprotein B - Precision (< 6.5 % Total CV)


Complement C3 - Precision (< 3.9 % Total CV)Level N Mean

mg/dLWithin

Run SDWithin Run

%CV Total SD

Total %CV


Level N Mean mg/dL

Within Run SD

Within Run %CV

Total SD

Total %CV


Complement C4 - Precision (< 4.5 % Total CV)


Haptoglobin - Precision (< 6.0 % Total CV)

Level N Mean mg/dL

Within Run SD

Within Run %CV

Total SD

Total %CV


Immunoglobulin A - Precision (< 4.1 % Total CV)Level N Mean

mg/dLWithin

Run SDWithin Run

%CV Total SD

Total %CV



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



Prealbumin - Precision (< 5.5 % Total CV)Level N Mean

mg/dLWithin

Run SDWithin Run

%CV Total SD

Total %CV


Transferrin - Precision (< 5.0 % Total CV)Level N Mean

mg/dLWithin

Run SDWithin Run

%CV Total SD

Total %CV



Method Comparison


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


Method Comparison


-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


AEROSET®


AEROSET®

vs. Hitachi

Complement C4Complement C3


Method Comparison


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


AEROSET®


AEROSET®

vs. Hitachi

Immunoglobulin AHaptoglobin


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


AEROSET®


AEROSET®

vs. Hitachi

Immunoglobulin MImmunoglobulin G


Method Comparison


-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


AEROSET®


AEROSET®

vs. Hitachi

TransferrinPrealbumin


Assay Specific Information


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


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)







1:4 Dil 1 protocol addedLH field contains lowest possible value for high calibrator


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)







Reference ranges adjusted to reflect recent literatureCalibration interval extended to 57 daysLH field contains lowest possible value for high calibrator


Complement C4Assay Method: ImmunoturbidimetricReagent List#: 9D97-20Calibraton:


Assay Stabilityonboard Stability: 57 Days (1368 hours)Calibration Stability: 57 Days(1368 Hours)


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


HaptoglobinAssay Method:

ImmunoturbidimetricReagent List#: 9D91-20Calibraton:


Assay Stabilityonboard Stability: 57 Days(1368 hours)Calibration Stability: 57 Days(1368 Hours)







New Information1:4 Dil 2 protocol addedCal interval extended to 57 daysLH field contains lowest possible value for high calibrator


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:


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 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


Immunoglobulin G (IgG)Reportable range:


Limit of Quantitation: < 61 mg/dL (0.61 g/L)Precision: < 3.4 % Total CVAcceptable specimen: plasma or serumAnticoagulants Tested:


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 Stabilityonboard Stability: 23 Days (552 hours)Calibration Stability: 23 Days (552 hours)

Assay VolumesSpecimen: 2.7 uLR1 Reagent: 160 uLR2 Reagent: 160 uL


Immunoglobulin M (IgM)Reportable range:



Precision: < 4.4 % Total CVAcceptable specimen: plasma or serumAnticoagulants Tested:


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:


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


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)




Precision: < 5.5 % Total CVAcceptable specimen: serum

onlyNew Information

Calibration interval extended to 57 days


TransferrinAssay Method: ImmunoturbidimetricReagent List#: 1E04-20Calibraton:


Assay Stabilityonboard Stability: 57 Days(1368 hours)Calibration Stability: 57 Days(1368 Hours)







New InformationReference ranges adjusted to reflect recent literatureCalibration interval extended to 57 days


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



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)



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.



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


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.


Questions and Answers


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.



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.


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.



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.



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.



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



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



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.



• 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.


Any Questions??????

End of Document

clinical chemistry serum proteins - yeec · 201020-101 clinical chemistry specific proteins...

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