Procedure: FERRITIN OSR61203 This procedure is valid for the following chemistry analyzers:

AU400/AU400e AU480 AU600 AU5400

AU640/AU640e AU680 AU2700 AU5800

Prepared By

Date Adopted

Supersedes Procedure #

Review Date

Revision Date

Signature

# of Distributed to Copies Distributed to

# of Copies

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Procedure: FERRITIN OSR61203

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Procedure: FERRITIN OSR61203

PRINCIPLE
Serum Ferritin is a sensitive indicator of body iron stores. It has been shown to correlate with stainable bone-marrow iron.1 Serum Ferritin is especially useful in distinguishing iron deficiency from the anemia of chronic disorders. A serum Ferritin of less than 10 ng/mL almost always indicates iron deficiency. Serum Ferritin is also increased in other anemias including aplastic anemia, sideroblastic anemia, and chronic hemolytic anemias. In idiopathic hemochromatosis and, in multiple transfused patients, the serum ferritin may be extremely high.1

IN!ENDED "SE
System reagent for the quantatitive determination of Ferritin in human serum on Beckman Coulter AU Clinical Chemistry analyzers.

#E!$%D%L%&'
Latex agglutination reactions occur as a result of antibody-coated latex beads aggregating if antigen is present in sufficient quantity. Immune complexes formed in solution scatter light in proportion to their size, shape and concentration. Under conditions of antibody excess, increasing amounts of antigen result in higher scatter. Turbidimeters measure the reduction of incident light due to reflection, absorption, or scatter. The Beckman Coulter AU procedure is based on the measurement of the decrease in light intensity transmitted (increase in absorbance) through particles suspended in solution as a result of complexes formed during the antigen-antibody reaction. The anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. When serum, containing ferritin, is mixed with the anti-ferritin reagent, an agglutination mixture occurs. This is measured spectrophotometrically on Beckman Coulter AU Chemistry Analyzers.

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Procedure: FERRITIN OSR61203

SPECI#EN
PA!IEN! PREPARA!I%N
None required.
Additional instructions for patient preparation as designated by this laboratory:

!'PE
Serum, Lithium heparin plasma, and EDTA plasma are the recommended samples. Strongly lipemic samples should be avoided.
Additional type conditions as designated by this laboratory:

$ANDLIN& C%NDI!I%NS
Serum and plasma samples are stable for 7 days when stored at 2-8 C and for 12 months when frozen (< -20 C).2

Additional handling conditions as designated by this laboratory:

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Procedure: FERRITIN OSR61203

E("IP#EN! AND #A!ERIALS

E("IP#EN!
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, AU5400 and AU5800 analyzers.

#A!ERIALS
Beckman Coulter AU System Ferritin Reagents. Final concentration of reactive ingredients: )l*c!ne $u%%er +R1, - ..2/ R2, - 0.31 Also contains preservatives.
Reagent storage location in this laboratory:

100

mmol2L

Latex art!cle# coate" 3!th ra$$!t ant!4human %err!t!n

Test tubes 12 -16 mm in diameter or sample cups

Storage location of test tubes or sample cups in this laboratory:

(Cat No. AU1063).

Beckman Coulter Serum Protein Multi-Calibrator (Cat. No. ODR3021).

Storage location of the Calibrator in this laboratory:

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Procedure: FERRITIN OSR61203 Precautions: 1. For in vitro diagnostic use. 2. Do not ingest reagent or calibrator. Harmful if swallowed. 3. Contains sodium azide as a preservative that may react with lead joints in copper plumbing to form explosive compounds. Even though the reagent contains minute quantities of sodium azide, drains should be well flushed with water when discarding the reagent or calibrators. 4. WARNING: POTENTIAL BIOHAZARDOUS MATERIAL. The calibrator is manufactured from human serum. No test method can offer complete assurance that HIV- 1/2, HCV, Hepatitis B, or other infectious agents are absent from biological materials, all calibrator material should be handled at the Biosafety Level 2 as recommended for any infectious human serum or blood specimen in the CDC/National Institutes of Health manual, Biosafety in Microbiological and Biomedical Laboratories, 1993. 5. Dispose of all waste material in accordance with local guidelines.

PREPARA!I%N
The Beckman Coulter AU System Ferritin reagent is liquid, ready to use. No preparation is needed. The R2 latex solution should be mixed by inversion 5 - 10 times before placing on the instrument and at weekly intervals thereafter. The Beckman Coulter Serum Protein Multi-Calibrators (Cat No. ODR3021) are liquid, ready for use as supplied. Mix by gentle inversion to achieve a homogenous mixture prior to use. Storage Requirements: 1. The unopened reagents and calibrators are stable until the expiration date printed on the label when stored at 2 - 8C.

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Procedure: FERRITIN OSR61203 2. Opened bottles of reagent are stable for 60 days when stored in the refrigerated compartment of the Beckman Coulter AU analyzers. Mix the R2 latex reagent at weekly intervals as described in Preparation section above. 3. Opened bottles of calibrator are stable for 30 days provided stoppers and caps are replaced immediately after each use. The Multi-calibrators should be stored at 2 - 8C when not in use.

Additional storage requirements as designated by this laboratory:

Indications of Deterioration: Discoloration of the reagent or calibrators, visible signs of microbial growth, turbidity or precipitation in reagent or calibrators may indicate degradation and warrant discontinuance of use.

PER)%R#ANCE PARA#E!ERS
The following data was obtained using the Ferritin Reagent on an Beckman Coulter AU Analyzers according to established procedures. Results obtained in individual laboratories may differ.

PRECISI%N

*+

Estimates of precision, based on CLSI recommendations11, are consistent with typical performance. The within run precision is less than 8%CV and total precision is less than 11%CV. Assays of control sera were carried out and data reduced following CLSI guidelines.
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Procedure: FERRITIN OSR61203

N,-+ #ean1 ng2dL SD .it/in run C34 SD !ota0 C34

25 1(0 (30

1.15 1.0. 1..6

(.0 0.0( 0.(3

1.21 1..1 (.36

(.62 1.23 1.0

The above data based on 3 serum pools analyzed over 20 days on AU640.

C%#PARIS%N

*+

Patient samples were used to compare the Beckman Coulter AU System Ferritin Reagent. Representative performance data on AU analyzers is shown in the next table.

' #et/od 7 #et/od S0ope Intercept Corre0ation Coeff9 :r; No9 of Sa<p0es :n; Range :ng2dL;

A"56+2A"56+e #et/od 8 1.06 (.3 0.662 103 6.0 7 ((0.6

Analytical Sensitivity (Lower Detection Limit) The lowest detectable level on an AU640 analyzer was calculated as 2.8 ng/mL. The lowest detectable level represents the lowest measurable level of Ferritin that can be distinguished from zero. It is calculated as the absolute mean plus three standard deviations of 20 replicates of an analyte free sample.

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Procedure: FERRITIN OSR61203 Sensitivity: The Limit of Detection (LOD) for this Ferritin reagent was determined to be less than 4.3 ng/mL. This was determined according to CLSI protocol EP17-A.12 Limit of Quantitation: The Limit of Quantitation (LOQ) for the Ferritin reagent was determined to be less than 7.8 ng/mL. This was determined according to CLSI protocol EP17-A12 and represents the lowest concentration of Ferritin that can be measured with a total imprecision of 20%.

CALIBRA!I%N
S!ANDARD PREPARA!I%N
Perform a multi-point calibration (5AB) using a water blank (blue rack) and the appropriate calibrators in a yellow calibration rack. The frequency of calibration is every 30 days. Calibration of this Ferritin procedure is accomplished by use of the Beckman Coulter Serum Protein Multi-Calibrator (Cat No. ODR3021), which is traceable to the 3 rd International Standard for Ferritin, Recombinant NIBSC code: 94/572. The Serum Protein Multi-Calibrator is a 5 - level calibrator for Ferritin. This calibrator has not been tested for use with any other Chemistry Systems other than those listed above. The results obtained using this calibrator are dependent upon several factors, including proper storage of the calibrator and proper technique in use of the Beckman Coulter AU Clinical Chemistry analyzers and their respective reagents.

CALIBRA!I%N PR%CED"RE
Recalibration is required when any of the following conditions occur:

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Procedure: FERRITIN OSR61203 1. A reagent lot number has changed or there is an observed shift in control values. 2. Major preventative maintenance was performed on the analyzer. 3. A critical part was replaced. Following calibration, the resulting calibration curve should be visually reviewed, on the AU analyzer for acceptability.

("ALI!' C%N!R%L
During operation of the Beckman Coulter AU analyzer at least two levels of an appropriate control material should be tested a minimum of once a day. In addition, these controls should be performed after calibration, with each new lot of reagents, and after specific maintenance or troubleshooting steps described in the appropriate AU Users Guide. Quality control testing should be performed in accordance with regulatory requirements and each laboratorys standard procedure.

Location of controls used at this laboratory.

ANAL'=ER PARA#E!ERS
A complete list of test parameters and operating procedures can be found in the appropriate Users Guide and at www.beckmancoulter.com.

CALC"LA!I%NS
Values set for working in SI Units g/L, equivalent to ng/mL.

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Procedure: FERRITIN OSR61203

REP%R!IN& RES"L!S
RE)ERENCE RAN&ES
Male1: Female1:
-1>

16 - 243 ng/mL 10 - 158 ng/mL

Expected values may vary with age, sex, diet and geographical location. Each laboratory should determine its own expected values as dictated by good laboratory practice.
Expected reference ranges in this laboratory:

PR%CED"RES )%R ABN%R#AL RES"L!S

Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the instrument parameters.

REP%R!IN& )%R#A!
Results are automatically printed out for each sample in ng/mL at 37 C.
Additional reporting information as designated by this laboratory:
o

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Procedure: FERRITIN OSR61203

LI#I!A!I%NS
The Beckman Coulter AU System Ferritin assay is linear from 8 - 450 ng/mL. Samples exceeding the upper limit of linearity should be diluted and repeated. The sample may be diluted, repeated and multiplied by the dilution factor automatically utilizing the AUTO REPEAT RUN. Prozone or hook effect may occur with highly elevated Ferritin samples (>20,000 ng/mL) This assay has been specifically designed to substantially reduce the risk of interference from HAMA or heterophilic antibodies. However, as with all immunoassays there is always a small risk from such interferences. Therefore, for diagnostic purposes the Ferritin results should always be assessed in conjunction with other available information, e.g. patient medical history, clinical impressions, and results of other clinical tests.5,6,7 Investigation of samples believed to contain interferences can take a number of approaches. Serial dilution may reveal incorrect recovery. PEG precipitation, pre-treatment with non-immune serum, and assay of the sample in an alternate assay system are also useful in identifying whether the samples contains interferent. The results of such samples should be interpreted with extreme care.7 Samples with extremely abnormal optical characteristics, especially turbidity, may produce atypical results. Such samples must be serially diluted and repeat results compared to ensure that no such interference exists.

IN!ER)ERIN& S"BS!ANCES
Results of studies3 conducted show that the following substances interfere with this Ferritin procedure: The criteria for no significant interference is recovery within 10% of the initial value. B!l!ru$!n, -emol*#!#, L! em!a, 8o #!&n!%!cant !nter%erence u to (0 m&2"L B!l!ru$!n 8o #!&n!%!cant !nter%erence u to 500 m&2"L -emol*#ate 8o #!&n!%!cant !nter%erence u to (00 m&2"L Intral! !"9

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Rheumato!" %actor,

Procedure: FERRITIN OSR61203 8o #!&n!%!cant !nter%erence u to 500 I:.mL R;.

* Intralipid, manufactured by KabiVitrium Inc., is a 20% IV fat emulsion used to emulate extremely turbid samples. In very rare cases gammopathy, especially monoclonal IgM (Waldenstrms macroglobulinemia), may cause unreliable results. The information presented is based on results from Beckman Coulter AU studies and is current at the date of publication. Beckman Coulter, Inc., makes no representation about the completeness or accuracy of results generated by future studies. For further information on interfering substances, refer to Young4 for a compilation of reported interferences with this test.

Laboratory specific procedure notes:

RE)ERENCES
1. Henry, J.B., [ed] Clinical Diagnosis and Management by Laboratory Methods, 19th Edition, Philadelphia: WB Saunders, 1996. 2. WHO, Use of anticoagulants in diagnostic laboratory investigations and stability of blood, plasma, and serum samples.WHO/DIL/LAB/99.1 rev 2, 2002:31. 3. CLSI, Interference testing in clinical chemistry, EP7-A2, 2005. 4. Young, D.S., Effects of Drugs on Clinical Laboratory Tests, AACC, Fifth Edition, AACC Press, 2000.
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Procedure: FERRITIN OSR61203 5. Selby C. Interference in Immunoassay. Ann Clin Biochem 1999;36:704 721. 6. Ismail AA. On the interpretation of affirmative follow-up tests in immunoassays; what must not be done? Ann Clin Biochem 2006; 43(4): 249-251. 7. CLSI, Immunoassay interference by endogenous antibodies, proposed guideline, I/LA30-P, 2007. 8. Data is on file at Beckman Coulter, Inc. 9. CLSI, Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline. C28-A3, 2008. 10. Data is on file for specific AU chemistry analyzers. 11. CLSI, Evaluation of precision performance of quantitative measurement methods, EP5-A2; 2004. 12. CLSI, Protocol for determination of limit of detection and limit of quantitation; Approved guideline, EP17-A, 2004.

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