Instructions for Use

2008-03

Apolipoprotein B (APO B)
System New Information

ADVIA® 1200 Updates to Reagents, Reagent

Preparation and Use, Calibration,
Calibration Frequency, Quality Control,
References, and Trademarks
ADVIA 1650/1800 Updates to Reagents, Reagent
Preparation and Use, Calibration,
Calibration Frequency, Quality Control,
References, and Trademarks
ADVIA 2400 Updates to Reagents, Reagent
Preparation and Use, Calibration,
Calibration Frequency, Quality Control,
References, and Trademarks

NOTE: For an explanation of the symbols in this document and associated products,
refer to Understanding the Symbols located on the introductory page of the Methods
Directory.

Method Summary
Item Description
Method Principle PEG enhanced immunoturbidimetric
Specimen Type Human serum and plasma (lithium heparin)
On-board Stability ADVIA 1200: 30 days
ADVIA 1650/1800: 30 days
ADVIA 2400: 30 days
Reagent Storage Temperature 2 – 8°C
Calibration Frequency ADVIA 1200: 30 days
ADVIA 1650/1800: 30 days
ADVIA 2400: 30 days
Reaction Blank (RBL) Frequency Daily
Reaction Type Endpoint (EPA)
Measurement Wavelength 340/694 nm
Standardization WHO-IFCC reference material SP3-07

© 2007 Siemens Medical Solutions Diagnostics. All rights reserved.

Siemens Medical Solutions Diagnostics, Tarrytown, NY 10591-5097 USA
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Apolipoprotein B (APO B)

Item Description
Analytical Range System Serum/Plasma*
ADVIA 1200 1 – (200 – 260 mg/dL)
(0.01 – [2.00 – 2.60 ] g/L)
ADVIA 1650/1800 15 – (200 – 260 mg/dL)
(0.15 – [2.00 – 2.60] g/L)
ADVIA 2400 15 – (200 – 260 mg/dL)
(0.15 – [2.00 – 2.60] g/L)
*the APOB concentration in the ADVIA Chemistry
Apolipoprotein A1/B Calibrator Level 5 varies from
200 − 260 mg/dL (2.00 − 2.60 g/L)
Expected Values Male: 46 – 174 mg/dL
(0.46 – 1.74 g/L)
Female: 46 – 142 mg/dL
(0.46 – 1.42 g/L)
Reagent Code 74086

Item Description
Calibrator ADVIA Chemistry Apolipoprotein A1/B Calibrator
REF 02269277 (PN B03-4161-01)
Serum Controls ADVIA Chemistry Lipid Controls:
Level 1 REF 01313493 (PN B03-4766-01)
Level 2 REF 06413232 (PN B03-4767-01)

Intended Use
For in vitro diagnostic use in the quantitative determination of apolipoprotein B in
human serum and plasma on the ADVIA Chemistry systems. Such measurements are
used to aid in the assessment of risk for arteriosclerosis and coronary artery disease.

Summary and Explanation 1-4

Apolipoprotein B (APO B) is a distinct, lipid-binding protein that is the major protein
moiety of all lipoproteins other than high-density lipoprotein (HDL).1-3 APO B is
essential to the formation and release of lipoproteins into the plasma and interacts with
the LDL receptors of peripheral cells thereby functioning in the recognition of cellular
receptors for the catabolism of LDL.2-3

Studies show a correlation between the development of coronary atherosclerosis and

abnormal concentrations of lipoproteins. In atherosclerosis, the inner layer of the
arterial wall thickens due to the accumulation of cellular material and deposits of
several substances, particularly lipids.3 Because apolipoproteins are unique markers
for the identification and differentiation of lipoproteins, they are the most probable
determinants of the structural integrity and functional specificity of lipoproteins.1
Measurement of APO B in serum of patients with significant arteriosclerotic alterations
shows APO B levels significantly higher than a normal patient population.4 Abnormal
APO B values also occur with dyslipoproteinemias. Adult-onset diabetes, and liver
diseases including acute hepatitis and fatty liver result in elevated APO B values.1

Numerous studies suggest that serum Apolipoprotein A-1(APO A-1) and APO B levels
indicate the severity and extent of coronary artery stenosis better than serum total

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Apolipoprotein B (APO B)

cholesterol and triglyceride.2 In addition, studies indicate that patients with

atherosclerosis are better distinguished from patients without the disease by the finding
of increased plasma APO B levels than by the findings of decreased HDL-C and
increased LDL-C levels.3 Measuring both APO A-1 and APO B and expressing the data
as a ratio has been shown to be more effective than APO A-1 or APO B alone.2

Principles of the Procedure

The APO B method is a polyethylene glycol (PEG) enhanced immunoturbidimetric
assay. A sample containing human apolipoprotein B and specific antiserum form an
insoluble complex that is measured turbidimetrically at 340/694 nm. By constructing a
standard curve from the absorbances of standards, the concentration of
apolipoprotein B is determined.

Reagents
The reagents are packaged as listed below. Components of the package are available
as a kit only.

REF (PN) Symbol Contents Amount No. of Tests

Container Size
03055068 Apolipoprotein B 2 x 85
Reagents
20-mL Reagent 1 2 x 10.0 mL

20-mL Reagent 2 2 x 4.0 mL

Components and Concentrations

Reagent Component Concentration

Reagent 1 Polyethylene glycol 4%
Sodium azide 0.09%
Reagent 2 Antihuman APO B (sheep) lot-specific
Sodium azide 0.09%

CAUTION! This device contains material of animal origin and should be handled as a
potential carrier and transmitter of disease.

NOTE: Sodium azide can react with copper and lead plumbing to form explosive metal
azides. If disposal into a drain is in compliance with federal, state, and local
requirements, flush reagents with a large amount of water to prevent the buildup of
azides.

For In Vitro Diagnostic Use.

Reagent Preparation and Use

Reagents are ready to use. Before use, gently swirl the reagent to disrupt bubbles and
assure homogeneity. If bubbles still exist or foam is present, using a clean transfer
pipette, aspirate them from the reagent container prior to use.

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Apolipoprotein B (APO B)

The ADVIA 1200 Chemistry system automatically predilutes serum and plasma
samples. Place a 70-mL reagent container (REF 06397121; PN 073-0373-01) filled
with saline (0.9% NaCl) into RTT 1, position 1. Refill this reagent container once per
shift, or as needed. Remove the container from the system weekly and clean or replace
it with a new container.

On-board Reagent Stability (OBS)

System Stability
ADVIA 1200 30 days
ADVIA 1650/1800 30 days
ADVIA 2400 30 days

For all systems, unopened reagents are stable until the expiration date printed on the
product label when stored at 2° – 8°C. Do not freeze reagents.

For additional details, refer to the Methods Introduction section of the system-specific
Operator’s Guide.

Sample Handling
For additional details, refer to Sample Collection and Preparation in the Methods
Introduction section of the system-specific Operator’s Guide.

For instructions on how to load reagents and run samples, refer to the Daily Operations
section of the system-specific Operator’s Guide.

Materials Required but not Provided

The following list contains the materials required, but not provided, to perform this
method:
• sample containers
• system solutions
• calibrator and control materials (refer to the Summary section for the REFs)
• 70-mL reagent container (REF 06397121; PN 073-0373-02)
• reagent container adapter:
• 20-mL adapter (REF 02404085; PN 094-0159-01) for 40-mL slot
(ADVIA 1200/1800)
• 20-mL adapter (REF 05249323; PN 073-0936-01) for 70-mL slot (ADVIA 1800)
• 20-mL adapter (REF 00771668; PN 073-0345-02) for 70-mL slot
(ADVIA1650/2400)

For storage and stability information, refer to the package insert.

Calibration
Refer to the package insert supplied with the ADVIA Chemistry Apolipoprotien A1/B
Calibrator (REF 02269270; PN B03-4161-01) for handling instructions and values. For
setup and use instructions, refer to the Calibration Overview section of the
system-specific Operator’s Guide.

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Apolipoprotein B (APO B)

Reconstituting the ADVIA Chemistry Apolipoprotein A1/B

Calibrator for ADVIA 1650/1800/2400 Systems
1. Reconstitute the Apolipoprotein A1/B Calibrator according to the package insert
instructions.
2. Manually dilute the reconstituted Apolipoprotein A1/B Calibrator to a 1:4 dilution
(1 part calibrator with 3 parts isotonic saline).
3. Place 1 sample container of the 1:4 diluted Apolipoprotein A1/B Calibrator onto the
sample tray (STT), at the cup position assigned to the calibrator.
4. Place another sample container with saline at the cup position assigned to the
blank for calibration.

Reconstituting the ADVIA Chemistry Apolipoprotein A1/B

Calibrator for ADVIA 1200 Systems
1. Reconstitute the Apolipoprotein A1/B Calibrator according to the package insert
instructions. No further dilution for the reconstituted calibrator is needed.
2. Place one sample container of the reconstituted Apolipoprotein A1/B Calibrator
onto the sample tray (STT), at the cup position assigned to the calibrator for
calibration.
3. Place another sample container with saline at the cup position assigned to the
blank for calibration.

The ADVIA Chemistry systems automatically dilute the Apolipoprotin A1/B calibrator
placed in the STT to obtain each of the 4 standard levels required for the multiple-level
standard calibration for this method.

Calibration Frequency
Perform a calibration when this method is implemented on the system. You must
recalibrate after the following events:
• when the reagent lot number changes
• after replacing critical optical or hydraulic components
• when indicated by quality control procedures

Siemens has validated the calibration stability for this method as shown in the following
table:

Minimum Calibration
System Stability*
ADVIA 1200 30 days
ADVIA 1650/1800 30 days
ADVIA 2400 30 days
*or whenever indicated by quality control data

Siemens recommends calibrating new reagent packs if the previous reagent pack was
calibrated any time during its on-board stability, other than as a fresh pack.

Reagent Blank (RBL) Frequency

The RBL is measured daily with saline.

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Apolipoprotein B (APO B)

Quality Control
Siemens recommends the use of quality control material from Bio-Rad Laboratories
with at least 2 levels (low and high). A satisfactory level of performance is achieved
when the analyte values obtained are within the Acceptable Control Range for the
system or within your range, as determined by an appropriate internal laboratory
quality control scheme.

The actual frequency of control in a laboratory is based on many factors, such as

workflow, system experience, and government regulation. Each laboratory should
evaluate the controls based on the frequency established by their laboratory
guidelines. When the method is performed, analyze at least 2 levels of controls daily.

Also, assay controls under the following conditions:

• whenever you use a new reagent lot
• following the performance of any system maintenance, cleaning, or troubleshooting
procedure
• after performing a new calibration

For more information, refer to the Quality Control Overview section of the
system-specific Operator’s Guide.

Limitations of the Procedure 5

A number of substances cause physiological changes in serum or plasma analyte
concentrations. A comprehensive discussion of these and other interfering substances,
their serum or plasma concentrations, and their possible physiological involvements is
beyond the scope of this document. Consult listed reference for specific details on
known potential interfering substances.5

As with any chemical reaction, you must be alert to the possible effect on results of
unknown interferences from medications or endogenous substances. The laboratory
and physician must evaluate all patient results in light of the total clinical status of the
patient.

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Apolipoprotein B (APO B)

Interferences
Siemens tested the following interferents up to the indicated levels and found they did
not significantly interfere with this method:

ADVIA 1200
APO B Sample
Interferent Interferent Level Concentration Interference*
Bilirubin 60 mg/dL 95 mg/dL NSI
(conjugated/unconjugated) (1026 µmol/L) (0.95 g/L)
Hemolysis 1000 mg/dL 95 mg/dL NSI
(hemoglobin) (10.0 g/L) (0.95 g/L)
Lipemia 1000 mg/dL 95 mg/dL NSI
(from Intralipid) (11.3 mmol/L)** (0.95 g/L)
*NSI = No Significant Interference. A percentage effect ≥ 10% is considered a significant
interference.
**as triolein

ADVIA 1650/1800
APO B Sample
Interferent Interferent Level Concentration Interference*
Bilirubin 30 mg/dL 46 mg/dL NSI
(conjugated/unconjugated) (513 µmol/L) (0.46 g/L)
Hemolysis 525 mg/dL 46 mg/dL NSI
(hemoglobin) (5.25 g/L) (0.46 g/L)
Lipemia 650 mg/dL 46 mg/dL NSI
(from Intralipid) (7.35 mmol/L)** (0.46 g/L)
*NSI = No Significant Interference. A percentage effect ≥ 10% is considered a significant
interference.
**as triolein

ADVIA 2400
APO B Sample
Interferent Interferent Level Concentration Interference*
Bilirubin 30 mg/dL 95 mg/dL NSI
(conjugated/unconjugated) (513 µmol/L) (0.95 g/L)
Hemolysis 525 mg/dL 105 mg/dL NSI
(hemoglobin) (5.25 g/L) (1.05 g/L)
Lipemia 650 mg/dL 100 mg/dL NSI
(from Intralipid) (7.35 mmol/L)** (1.00) g/L
*NSI = No Significant Interference. A percentage effect ≥ 10% is considered a significant
interference.
**as triolein

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Apolipoprotein B (APO B)

Performance Characteristics
Precision 6
Each sample was assayed 2 times per run, 2 runs per day, for at least:
• 10 days for the ADVIA 1200
• 15 days for the ADVIA 2400
• 20 days for the ADVIA 1650/1800

Precision estimates were computed according to CLSI document EP05-A2, Evaluation

of Precision Performance of Quantitative Measurement Methods; Approved Guideline.6

Data contained in this section represents typical performance for

ADVIA Chemistry systems. Your laboratory data may differ from these values.

Conversion factor: mg/dL x 0.01 = g/L

ADVIA 1200
Within-Run Total
Specimen Type Level SD CV (%) SD CV (%)
Common Units (mg/dL)
Serum 113 1.4 1.2 5.9 5.2
Serum 150 1.8 1.2 7.2 4.8
SI Units (g/L)
Serum 1.13 0.014 1.2 0.059 5.2
Serum 1.50 0.018 1.2 0.072 4.8

ADVIA 1650/1800
Within-Run Total
Specimen Type Level SD CV (%) SD CV (%)

Common Units (mg/dL)

Serum 29 0.5 1.9 1.7 5.7
Serum 111 1.3 1.1 3.7 3.3
SI Units (g/L)
Serum 0.29 0.005 1.9 0.017 5.7
Serum 1.11 0.013 1.1 0.037 3.3

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Apolipoprotein B (APO B)

ADVIA 2400
Within-Run Total
Specimen Type Level SD CV (%) SD CV (%)

Common Units (mg/dL)

Serum 83 1.3 1.6 2.7 3.3
Serum 108 1.2 1.1 3.9 3.6
Serum 145 2.0 1.4 3.7 2.6
SI Units (g/L)
Serum 0.83 0.013 1.6 0.027 3.3
Serum 1.08 0.012 1.1 0.039 3.6
Serum 1.45 0.020 1.4 0.037 2.6

Analytical Range
This method measures the APO B concentration in serum and plasma ranging from the
minimum detectable concentration (MDC) to the APO B concentration in the highest
level of the calibrator according to the table shown below. MDC is an estimation based
on 2 times the within-run standard deviation of the zero calibrator.

System Serum/Plasma*
ADVIA 1200 1 – (200 – 260) mg/dL
(0.01 – [2.00 – 2.60] g/L)
ADVIA 1650/1800 15 – (200 – 260) mg/dL
(0.15 – [2.00 – 2.60] g/L)
ADVIA 2400 15 – (200 – 260) mg/dL
(0.15 – [2.00 – 2.60] g/L)
*the APOB concentration in the ADVIA Chemistry Apolipoprotein A1/B
Calibrator Level 5 varies from 200 − 260 mg/dL (2.00 − 2.60 g/L)

Siemens has validated automatic rerun conditions for this method that extend the
reportable range up to 400 mg/dL (4.0 g/L) on the chemistry systems for serum and
plasma.

Prozone Effect
A prozone effect was not demonstrated for APO B concentration up to 5600 mg/dL
(56.00 g/L).

Expected Values 7,8

According to published studies such as Clinical Guide to Laboratory Test, 3rd edition,
by NW Tietz and Lipoprotein Cholesterol, and Apoprotein Distribution in Selected US
Communities by SA Brown, et al. expected values vary according to age, sex, and
race.

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Apolipoprotein B (APO B)

The following table lists the reference ranges for this method:

Sex Reference Range

Male (Adult) 46 – 174 mg/dL
(0.46 – 1.74 g/L)
Female (Adult) 46 – 142 mg/dL
(0.46 – 1.42 g/L)

The above APO B reference range, which represent ranges seen in selected U.S.
populations, should be used as a rough guide in evaluating the degree of abnormality.7

Siemens provides these ranges for reference. Each laboratory should establish its own
normal range. You can enter normal range values and abnormal range values in the
Analytical Parameters (Chemistry) window.

System Correlation
The performance of the applicable method (y) was compared with the performance of
the same method on the comparison system (x).

ADVIA 1200
Specimen Type Comparison System (x) N Regression Equation Sy.x r Sample Range
Serum ADVIA 1650 119 y = 1.00x + 0.3 2.5 0.997 23 – 138 mg/dL
y = 1.00x + 0.003 0.025 0.997 0.23 – 1.38 g/L
Plasma* ADVIA 1200 (serum) 119 y = 1.01x - 1.0 3.1 0.994 24 – 137 mg/dL
y = 1.01x - 0.010 0.031 0.994 0.24 – 1.37 g/L
*lithium heparin

ADVIA 1650/1800
Specimen Type Comparison System (x) N Regression Equation Sy.x r Sample Range
Serum Dade Behring 59 y = 1.03x + 0.5 3.7 0.993 42 – 173 mg/dL
Nephelometer
y = 1.03x + 0.005 0.037 0.993 0.42 – 1.73 g/L
Plasma* ADVIA 1650 (serum) 71 y = 1.00x - 1.7 1.7 0.997 50 – 158 mg/dL
y = 1.00x - 0.017 0.017 0.997 0.50 – 1.58 g/L
* lithium heparin

ADVIA 2400
Specimen Type Comparison System (x) N Regression Equation Sy.x r Sample Range
Serum ADVIA 1650 147 y = 0.92x + 5.9 2.6 0.996 30 – 222 mg/dL
y = 0.92x + 0.059 0.026 0.996 0.3 – 2.22 g/L
Plasma* ADVIA 2400 (serum) 43 y = 0.97x + 7.4 2.7 0.990 62 – 155 mg/dL
y = 0.97x + 0.074 0.027 0.990 0.62 – 1.55 g/L
*lithium heparin

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Apolipoprotein B (APO B)

Standardization
The ADVIA APO B method is traceable to the WHO-IFCC reference material SP3-07.
Assigned values of the ADVIA Chemistry Apolipoprotein A1/B Calibrator and the
ADVIA Chemistry Lipid Controls are traceable to this standardization.

References
1. Alaupovic P. Structure and function of plasma lipoproteins with particular regard to
hyperlipoproteinemias and atherosclerosis. Ann. Biol. Clin. 38:83-93 (1980).
2. Naito HK. The clinical significance of apolipoprotein measurements. J. Clin.
Immunoassay. 9:11-16 (1986).
3. Burtis CA, Ashwood ER. Tietz Fundamentals of Clinical Chemistry. 4th ed.
Philadelphia, PA: WB Saunders Company; 370-380 (1996).
4. Riesen WF, Mordansini R, Salzmann C, et al. Apoproteins and lipids as
discriminators of severity of coronary heart disease. Atherosclerosis. 37:157-162
(1980).
5. Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests. 3rd ed.
Washington: AACC Press (1990).
6. Clinical and Laboratory Standards Institute (formerly NCCLS). Evaluation of
Precision Performance of Quantitative Measurement Methods; Approved Guideline
- Second Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2004.
NCCLS Document EP05-A2.
7. Tietz NW. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia, PA: WB
Saunders Company; 68-71 (1995).
8. Brown SA, Hutchinson R, Morrisett J, Boerwinkle E, et al. Plasma Lipid, Lipoprotein
Cholesterol, and APOprotein Distribution in Selected US Communities.
Arteriosclerosis and Thrombosis. 13:1139-1158 (1993).

Technical Assistance
For customer support, please contact your local technical support provider or
distributor.

Trademarks
ADVIA is a trademark of Siemens Medical Solutions Diagnostics.
Dade Behring is a trademark of Dade Behring, Inc.
Intralipid is a trademark of Fresenius Kabi AB.

Siemens Medical Solutions

Diagnostics Pty Ltd
ABN 65 007 436 651
885 Mountain Highway
Bayswater Victoria 3153
Australia

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