IMMAGE® Immunochemistry Systems KAP

Chemistry Information Sheet Kappa Light Chain

© Copyright 2010 Beckman Coulter, Inc. REF 446440 (150 tests)

For In Vitro Diagnostic Use

ANNUAL REVIEW
Reviewed by: Date Reviewed by: Date

PRINCIPLE
INTENDED USE

KAP reagent, when used in conjunction with IMMAGE® Immunochemistry Systems and Calibrator 1, is intended for the
quantitative determination of kappa light chain (KAP) (free and bound) in human serum and urine by rate nephelometry.

CLINICAL SIGNIFICANCE

Measurements of kappa light chains are used in the diagnosis and treatment of patients with numerous illnesses including
severe liver and renal disease, multiple myeloma, and other disorders of blood proteins.
Should a paraprotein be identified in blood or urine or both, its heavy and light chains should be typed and the
concentrations of polyclonal IgG, IgA, and IgM determined. These studies confirm whether the spike on the
electrophoretic pattern is indeed a paraprotein, they help to decide the probable prognosis, and they show whether the
polyclonal immunoglobulins are so low that they leave a patient vulnerable to infections.1

METHODOLOGY

The KAP test measures the rate of increase in light scattered from particles suspended in solution as a result of complexes
formed during an antigen-antibody reaction.

CHEMICAL REACTION SCHEME

SPECIMEN
TYPE OF SPECIMEN

Serum and urine are the recommended specimens.

Serum
Serum samples should be collected in the manner routinely used for any clinical laboratory test.2 Freshly drawn serum
from a fasting individual is preferred.

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Urine
Urine samples should be collected without a preservative. Samples contaminated with blood are not recommended.
Centrifuge urine samples at 3,000 x g for 10 minutes prior to analysis to remove any cells or other debris.

SPECIMEN STORAGE AND STABILITY

Serum
1. Tubes of blood are to be kept closed at all times and in a vertical position. It is recommended that the serum be
physically separated from contact with cells within two hours from the time of collection.3
2. If serum samples are not assayed within 8 hours, samples should be stored at +2°C to +8°C. If samples are not
assayed within 72 hours, samples should be stored frozen at -15°C to -20°C. Frozen samples should be thawed
only once. Analyte deterioration may occur in samples that are repeatedly frozen and thawed.3

Urine
Urine samples may be stored at +2°C to +8°C for up to 72 hours. Frozen samples are not recommended.
Additional specimen storage and stability conditions as designated by this laboratory:

SAMPLE VOLUME

For sample volumes refer to the Sampling Template.

CRITERIA FOR UNACCEPTABLE SPECIMENS

Refer to the PROCEDURAL NOTES section of this chemistry information sheet.

Criteria for sample rejection as designated by this laboratory:

PATIENT PREPARATION

Special instructions for patient preparation as designated by this laboratory:

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SPECIMEN HANDLING

Special instructions for specimen handling as designated by this laboratory:

REAGENTS
CONTENTS

Each kit contains the following items:

KIT COMPONENTS QUANTITY

KAP Cartridge 1
Antibody
Antigen Excess Solution (AGXS)
Evaporation Caps 2
KAP Reagent Bar Code Card 1

INITIAL VOLUMES OF SAMPLE AND REAGENTS IN THE CUVETTE

Serum Urine
Sample Volume 0.11 µL 24 µL
Total Reagent Volume 344.89 µL 321 µL
Antibody 21 µL 21 µL
Buffer 1 300 µL 300 µL
Diluent 1 23.89 µL

REACTIVE INGREDIENTS

REAGENT CARTRIDGE CONSTITUENTS VOLUME

KAP Antibody (processed goat sera) 3.9 mL
KAP Antigen Excess Solution (processed diluted human 1.2 mL
serum)
Sodium Azide (used as a preservative) < 0.1% (w/w)
Also non-reactive chemicals necessary for optimal system performance.

CAUTION
Sodium azide preservative may form explosive compounds in metal drain lines. See
National Institute for Occupational Safety and Health Bulletin: Explosive Azide Hazards
(8/16/76).

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 CAUTION
Because this product is of human origin, it should be handled as though capable
of transmitting infectious diseases. Each serum or plasma donor unit used in the
preparation of this material was tested by United States Food and Drug Administration
(FDA) approved methods and found to be negative for antibodies to HIV and HCV and
nonreactive for HbsAg. Because no test method can offer complete assurance that
HIV, hepatitis B virus, and hepatitis C virus or other infectious agents are absent, this
material should be handled as though capable of transmitting infectious diseases. This
product may also contain other human source material for which there is no approved
test. The FDA recommends such samples to be handled as specified in Centers for
Disease Control’s Biosafety Level 2 guidelines.4

MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KIT

IMMAGE Immunochemistry Systems Wash Solution

IMMAGE Immunochemistry Systems Buffer 1
IMMAGE Immunochemistry Systems Diluent 1
Calibrator 1
Centrifuge capable of 3,000 x g
At least two levels of control material

REAGENT PREPARATION

1. Invert cartridge gently before removing screw caps.

2. Remove screw caps from reagent cartridges. Check each cartridge for bubbles and remove any bubbles present.
3. Place evaporation caps on both reagent cartridge compartments before loading the cartridge on the instrument.
See Appendices for evaporation cap directions.
4. Reagent cartridges should be stored upright and can be removed from the refrigerator and used immediately.
5. Mix all buffers and diluents thoroughly by inversion. Remove screw cap from container. Check each container for
bubbles and remove any bubbles present. Place evaporation cap on container before loading the container on the
instrument. See Appendices for evaporation cap directions.

ACCEPTABLE REAGENT PERFORMANCE

Acceptability of a reagent is determined from the successful performance of quality control testing, as defined in the
QUALITY CONTROL section of this chemistry information sheet.

REAGENT STORAGE AND STABILITY

Storage conditions other than those recommended may cause erroneous results.
Reagent Cartridges
1. Return all reagent cartridges to the refrigerator (+2°C to +8°C) upon completion of the daily workload.
2. The KAP reagents are stable for 30 days with the evaporation caps in place. Alternatively, reagent life can be
maximized by replacing evaporation caps with screw caps and storing at +2°C to +8°C upon completion of the
daily workload.
3. The KAP reagents are stable until the expiration date on the label if the reagents are stored at +2°C to +8°C with
the screw caps in place.
Diluent 1 and Buffer 1
1. Diluent 1 and Buffer 1 are stable on the system for 30 days with the evaporation caps in place.

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 2. Diluent 1 and Buffer 1 are stable until the expiration date on the label if they are stored at room temperature with
the screw caps in place.
Reagent storage location:

CALIBRATION
CALIBRATOR REQUIRED

Calibrator 1

CALIBRATOR PREPARATION

No preparation is required.

CALIBRATOR STORAGE AND STABILITY

Calibrator 1 is stable until the expiration date printed on the calibrator bottle if stored capped in the original container at
+2°C to +8°C.
Calibrator storage location:

CAUTION
Because this product is of human origin, it should be handled as though capable
of transmitting infectious diseases. Each serum or plasma donor unit used in the
preparation of this material was tested by United States Food and Drug Administration
(FDA) approved methods and found to be negative for antibodies to HIV and HCV and
nonreactive for HbsAg. Because no test method can offer complete assurance that
HIV, hepatitis B virus, and hepatitis C virus or other infectious agents are absent, this
material should be handled as though capable of transmitting infectious diseases. This
product may also contain other human source material for which there is no approved
test. The FDA recommends such samples to be handled as specified in Centers for
Disease Control’s Biosafety Level 2 guidelines.4

CALIBRATION INFORMATION

1. The IMMAGE® Immunochemistry Systems calibration is reagent lot specific.

2. The KAP reagent lot should be recalibrated when changing Buffer 1 lot or following specific part replacements or
maintenance procedures as defined in the IMMAGE Operations Manual.
3. The IMMAGE Immunochemistry System is designed for minimum calibration. Calibrations retained in system
memory should be monitored by the performance of quality control procedures on each day of testing.

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 4. Calibration for KAP is stable for 30 days.
5. The system will automatically perform a verification check during calibration and produce a calibration report. The
system will alert the operator of a failured calibration. An explanation of any accompanying error message can be
found in the TROUBLESHOOTING Section of the IMMAGE® Immunochemistry Systems Operations Manual.
6. Calibration verification information can be found in the CALIBRATION VERIFICATION section of the IMMAGE®
Immunochemistry Systems Chemistry Reference Manual.

TRACEABILITY

For Traceability information refer to the Calibrator instructions for use.

QUALITY CONTROL
It is recommended that at least two levels of control material, normal and abnormal, be analyzed daily. Refer to the
CALIBRATORS AND CONTROLS section of the IMMAGE® Immunochemistry Systems Chemistry Reference Manual,
for a list of Beckman Coulter controls. Controls should also be run with each new calibration, with a new lot of reagent
or buffer, and after specific maintenance or troubleshooting as detailed in the IMMAGE® Immunochemistry Systems
Operations Manual. More frequent use of controls or the use of additional controls is left to the discretion of the user
based on work load and work flow.
The following controls should be prepared and used in accordance with the package inserts. Discrepant quality control
results should be evaluated by your facility.

Table 1.0 Quality Control Material

CONTROL NAME SAMPLE TYPE STORAGE

TESTING PROCEDURE(S)
1. After setup, load reagents onto the system as directed in the IMMAGE Operations Manual.
2. Select chemistries to be calibrated, if necessary. Load bar coded calibrators, controls, and samples or program
and load non-bar coded controls and samples for analysis as directed in the IMMAGE Operations Manual .
3. Follow the protocols for system operation as directed in the IMMAGE Operations Manual.

CALCULATIONS
The IMMAGE Immunochemistry System will automatically calculate results.

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REPORTING RESULTS
ESTIMATION OF FREE KAPPA AND LAMBDA LIGHT CHAINS

Beckman Coulter‘s nephelometric KAP and LAM reagent antisera measure both free and bound light chains in serum
and urine. Standardization of these protein kits is based on the equivalent weight of the intact immunoglobulin molecules
(IgG + IgA + IgM = Kappa + Lambda). This standardization applies whether the antibody attaches to a free light chain or
to a light chain determinant of the intact immunoglobulin molecule. The resulting antigen-antibody complex is quantitated
with the reported value corresponding to the equivalent weight of the intact immunoglobulin molecules. Approximately
17% of the total mass of the intact immunoglobulin represents a single light chain. If the sample contains no intact
immunoglobulin (as determined by electrophoresis), multiply the light chain result by 0.17 to obtain the true concentration
of free light chain. If the sample contains a mixture of intact immunoglobulin plus free light chain, refer to References (5)
for a method to determine total free light chain.

REFERENCE INTERVALS

The reference interval values for human serum kappa light chains were established using an Array® 360 System,
for a population of 120 apparently healthy male and female adults from California and were verified on the IMMAGE
Immunochemistry System. The reference interval values for Kappa/Lambda ratio were established using the IMMAGE
Immunochemistry System for a population of 122 apparently healthy male and female adults from California. The
reference interval values for human kappa light chains in urine were established on the IMMAGE Immunochemistry
System using the KAP Test for a population of 123 apparently healthy male and female adults, Ames Multistix protein
negative, from California.*

Table 2.0 Reference intervalsa

SAMPLE TYPE REFERENCE INTERVALS KAPPA/LAMBDA RATIO
Beckman Coulter Serum 629 – 1,350 mg/dL 1.53 – 3.29
Urine < 1.85 mg/dL NAb
(in 94% of the population
tested)
a Each laboratory should establish its own reference interval(s) based on its patient population.
b NA = Not applicable.

SAMPLE TYPE REFERENCE INTERVALS KAPPA/LAMBDA RATIO

Laboratory

Refer to References (6,7,8,9) for guidelines on establishing laboratory-specific reference intervals.

Additional reporting information as designated by this laboratory:

UNITS AND CONVERSION FACTOR

Results for the KAP test are reported in default units of mg/dL. Metric conversion within the same unit category will occur
automatically if a new unit is selected. A conversion factor must be entered when selecting a unit category different from
the default.

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Refer to the System Setup section of the IMMAGE Operations Manual for more detailed information on units and
conversion factors.

PROCEDURAL NOTES
LIMITATIONS

Samples containing monoclonal kappa light chains (free and bound) may result in a condition of antigen excess and
artificially decreased values. Since the presence of an M-protein can normally be detected using protein electrophoresis,
the validity of immunochemical results should be determined by observing consistency with an electrophoretic pattern.
If a sample is in antigen excess at the starting dilution, reassay at the next higher dilution should provide a result more
consistent with the electrophoretic pattern.

INTERFERENCES

1. The following substances were tested for interference with this methodology:

Table 3.0 Interferences

SUBSTANCE SOURCE LEVEL TESTED OBSERVED EFFECT
Bilirubin Porcine 5 – 30 mg/dL None
Lipid Human Triglyceride 200 – 1,000 mg/dL Nonea
Hemoglobin Human 100 – 500 mg/dL None
a Quantitation of specific proteins by nephelometry may not be possible in lipemic sera due to the extreme light scattering properties of the
sample.

2. Nonspecific interference can occur between less dilute serum samples and polymer-enhanced buffer when off-line
dilutions less than 1:36 are assayed. No interference has been shown for urine samples assayed at these dilutions.
3. Dust particles or other particulate matter (i.e. debris and bacteria) in the reaction solution may result in extraneous
light-scattering signals, resulting in variable sample analysis.

PERFORMANCE CHARACTERISTICS
ANALYTIC RANGE

The KAP test is designed to detect concentrations of this analyte using an initial 1:216 serum sample dilution and
undiluted (neat) urine samples.

Table 4.0 Analytical Range

SAMPLE TYPE BECKMAN COULTER ANALYTICAL RANGE
Serum Initial: 400 – 4,400 mg/dL
Extended: 11.1 – 26,400 mg/dL
Urine Initial: 1.85 – 20.4 mg/dL
Extended: 1.85 – 26,400 mg/dL

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REPORTABLE RANGE (AS DETERMINED ON SITE):

Table 5.0 Reportable Range

SAMPLE TYPE LABORATORY REPORTABLE RANGE

Refer to the IMMAGE® Immunochemistry Systems Chemistry Reference Manual section on CALIBRATION
VERIFICATION, for more details on laboratory reportable range.

SENSITIVITY

Sensitivity is defined as the lowest measurable concentration which can be distinguished from zero with 95% confidence.
Sensitivity for kappa light chain in serum determination is 11.1 mg/dL and sensitivity for kappa light chain in urine
determination is 1.85 mg/dL.

EQUIVALENCY

Equivalency was assessed by Deming regression analysis of samples to an accepted clinical method. Values obtained
for KAP using the IMMAGE KAP test were compared to the values obtained using an Array® 360 System. Both normal
and abnormal samples with known positive light chains were included in the analysis.

Table 6.0 Equivalency Values

SERUM Array 360 SYSTEM URINE Array 360 SYSTEM
N 220 103
Slope 1.037 0.970
Intercept - 9.16 1.36
Mean (IMMAGE) 1,023 265
Mean (Array 360) 995.6 273
Correlation Coefficient (r) 0.987 0.980

The equivalency values were determined using patient serum samples ranging from 66.5 to 7,560 mg/dL and patient
urine samples ranging from 1.95 to 1,730 mg/dL. Refer to References (10,11) at the end of this chemistry information
sheet for guidelines on performing equivalency testing.

PRECISION

A properly operating IMMAGE® Immunochemistry Systems should exhibit imprecision values less than or equal to the
maximum performance limits listed below. Maximum performance limits were derived by an examination of the precision
of various methods, proficiency test summaries, and literature sources.

Table 7.0 Maximum Performance Limits

CHANGEOVER VALUE
TYPE OF PRECISION SAMPLE TYPE SD (mg/dL) % CV (mg/dL)a
Within-run Serum 40.0 4.0 1,000
Total Serum 40.0 6.0 667

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Table 7.0 Maximum Performance Limits, Continued
CHANGEOVER VALUE
TYPE OF PRECISION SAMPLE TYPE SD (mg/dL) % CV (mg/dL)a
Within-run Urine 0.37 4.0 9.25
Total Urine 0.37 6.0 6.17
a When the mean of the test precision data is less than or equal to the changeover value, compare the test SD to the SD guideline given above to
determine the acceptability of the precision testing. When the mean of the test precision data is greater than the changeover value, compare the
test % CV to the guideline given above to determine acceptability. Changeover value = (SD guideline/CV guideline) x 100.

Comparative serum performance data for the IMMAGE Immunochemistry System evaluated using the NCCLS Proposed
Guideline EP5-T2 appears in the table below.12 Each laboratory should characterize their own instrument performance
for comparison purposes.

Table 8.0 Typical Imprecision Values

TYPE OF Test Mean Value
PRECISION SAMPLE Data Pointsa (mg/dL) SD (mg/dL) % CV
Within-run Serum Level 1 80 540 15.6 2.9
Serum Level 2 80 1,113 24.1 2.2
Serum Level 3 80 2,377 61.2 2.6
Total Serum Level 1 80 540 17.4 3.2
Serum Level 2 80 1,113 30.9 2.8
Serum Level 3 80 2,377 70.9 3.0
a The serum point estimate is based on the data from 1 system, run for 20 days, 2 runs per day, 2 observations per run on an instrument operated
and maintained according to the manufacturer‘s instructions.

Comparative urine performance data for the IMMAGE Immunochemistry System evaluated using the NCCLS Proposed
Guideline EP10-T2 appears in the table below. Each laboratory should characterize their own instrument performance
for comparison purposes.13

Table 9.0 Typical Imprecision Values

TYPE OF Test Mean Value
PRECISION SAMPLE Data Pointsa (mg/dL) SD (mg/dL) % CV
Within-run Urine Level 1 30 2.33 0.059 2.5
Urine Level 2 30 9.34 0.236 2.5
Urine Level 3 30 19.2 0.25 1.3
Total Urine Level 1 30 2.33 0.081 3.5
Urine Level 2 30 9.34 0.314 3.4
Urine Level 3 30 19.2 0.56 2.9
a The urine point estimate is based on the data from 1 system, run for 5 days, 2 runs per day, 3 observations per run on an instrument operated
and maintained according to the manufacturer‘s instructions.

Refer to References (10,12,13) for guidelines on performing precision testing.

NOTICE
These degrees of precision were obtained in typical testing procedures and are not
intended to represent performance specifications for this test procedure.

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ADDITIONAL INFORMATION
For more information, refer to the IMMAGE Immunochemistry Systems Operations Manual.

SHIPPING DAMAGE

If damaged product is received, notify your Beckman Coulter Clinical Support Center.

FOOTNOTES

* Multistix is a registered trademark of Ames.

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REFERENCES
1. Burtis, C. A., Ashwood, E. R., Tietz Textbook of Clinical Chemistry, 2nd Edition, W. B. Saunders, Philadelphia, PA
(1994).

2. Tietz, N. W., "Specimen Collection and Processing; Sources of Biological Variation", Textbook of Clinical
Chemistry, pp 478 518, W. B. Saunders, Philadelphia, PA (1986).

3. National Committee for Clinical Laboratory Standards, Procedures for the Handling and Processing of Blood
Specimens, Approved Guideline, NCCLS publication H18-A, Villanova, PA (1990).

4. CDC-NIH manual, Biosafety in Microbiological and Biomedical Laboratories, U.S. Government Printing Office,
Washington, D.C. (1984).

5. Beckman Special Chemistry Monograph, "Kappa and Lambda Light Chain Measurements", K/L 1.

6. National Committee for Clinical Laboratory Standards, How to Define, Determine, and Utilize Reference Intervals
in the Clinical Laboratory, Approved Guideline, NCCLS publication C28-A, Villanova, PA (1992).

7. Tietz, N. W., Clinical Guide to Laboratory Tests, 2nd Edition, W. B. Saunders, Philadelphia, PA (1990).

8. Henry, J. B., ed., Clinical Diagnosis and Management by Laboratory Methods, 17th Edition (1984).

9. Statland, Bernard E., "Clinical Decision Levels for Lab Tests", Medical Economic Book, Oradel, New Jersey (1983).

10. Tietz, N. W., ed., Fundamentals of Clinical Chemistry, 3rd Edition, W. B. Saunders, Philadelphia, PA (1987).

11. National Committee for Clinical Laboratory Standards, Method Comparison and Bias Estimation Using Patient
Samples, Tentative Guideline, NCCLS publication EP9-T, Villanova, PA (1993).

12. National Committee for Clinical Laboratory Standards, Precision Performance of Clinical Chemistry Devices,
Tentative Guideline, 2nd Edition, NCCLS publication EP5-T2, Villanova, PA (1992).

13. National Committee for Clinical Laboratory Standards, Preliminary Evaluation of Quantitative Clinical Laboratory
Methods, Tentative Guideline, 2nd Edition, NCCLS publication EP10-T2, Villanova, PA (1993).

Beckman Coulter Ireland Inc., Mervue Business Park, Mervue, Galway, Ireland (353 91 774068)

Beckman Coulter, Inc., 250 South Kraemer Blvd., Brea, CA 92821

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