Ferritin Examination Procedure

1.Purpose of examination: Ferritin estimation from serum or plasma by CMIA technology (Chemiflex) Method.

2.Responsibility and Authority:

• Calibration: Technician
• Quality Control: Technician
• Routine operation: Technician
• Overall Monitoring: Quality Manager

3.Sample Details:

1. Type of Sample: Serum, Plasma
2. Type of container and additives: Plain without any additives
3. Patient Preparation: As per Primary Sample Collection Manual sample_collection_manual
4. Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
5. Handling and transport: As per Primary Sample collection manual
6. Storage: 24 hours at 2-8° C

4.Required Equipment: Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer

5.Required reagents: Microparticles: Anti-Ferritin (mouse, monoclonal) coated Microparticles in TRIS buffer with protein (mouse and bovine) stabilizers. Minimum Concentration: 0.125% solids. Conjugate : Anti-Ferritin (rabbit, polyclonal) acridinium labeled Conjugate in MES buffer with protein (bovine) stabilizers. Minimum concentration: 75 ng/mL. Multi Assay manual dilution : phosphate buffered saline solution. Pre trigger solution: 1.32% (w/v) hydrogen peroxide. Trigger solution : 0.35 N sodium hydroxide. Wash-buffer : phosphate buffered saline solution.

6.Reagent Handling Remove any air bubbles present in the reagents with a new applicator stick, or allow the reagents to settle at the appropriate storage temperature to allow the bubbles to dissipate. To minimize volume depletion, do not use a transfer pipette to remove bubbles

7.Reagent Storage and stability

• Unopened reagent stable at 15-30°C until expiration date.
• On board System temperature reagent is stable for 30 days
• Instability or deterioration should be suspected if there are precipitates, visible signs of leakage or
• contamination, turbidity, or if calibration or controls do not meet the appropriate criteria.

8.Calibration Procedure:

• Ferritin Calibrators
• Frequency:

1. Reagent lot change
2. QC out of range
3. After service or maintenance
4. Replacement in any parts of Instrument

Procedure:

1. Start the equipment.WDI abbotte fully.docx
2. Calibrators are ready to use.
3. Put calibrator 15-20 minutes at room temperature
4. Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation.
5. Take a 150 µl calibrator solution in to separate aliquots.
6. Go to the calibration and give the calibration order.
7. Verify calibration with at least two levels of controls
8. If control results fall outside acceptable ranges,root cause analysis or recalibration may be necessary.

9.Quality control Procedure: Name: Bio Rad Level 1 , 2 & 3 Frequency: As per Quality Control Procedure

• Procedure for Reconstitution of IQC
• Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
• Leave to stand for 30 min in the dark place.
• Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
• Prior to use, mix the contents by inverting the vial. Do not shake the vial as the information of foam should be avoided. Ensure that no lyophilized material remains un-reconstituted.
• Prepare aliquots of 150 µl from the reconstituted QC material.
• Store these aliquots at -15° C to -20° C.
• Prior to use, make sure that aliquots should be at room temperature for at least 15 min.

Procedure to run IQC

1. Press Control order
2. Select Assay /Panel, to be run.
3. Select the control/s and its level/s
4. Give Carrier Number and Position number
5. Press F3 / Add order
6. Put respected carrier in RSH rack
7. Check IQC results, in case outliers call residents.

10.Principle of the procedure used for examinations:

1. Sample, and anti-ferritin coated paramagnetic microparticles are combined. The ferritin present in the sample binds to the anti- ferritin coated microparticles.
2. After washing, anti-ferritin acridinium-labeled conjugate is added to create a reaction mixture.
3. Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction mixture.
4. The resulting chemiluminescent reaction is measured as relative light units (RLUs). There is a direct relationship between the amount of ferritin in the sample.

11.Sample Preparation: Required SampleVolume: 150 µl of the sample Temperature: 37° Take 150-200µl of the sample from Primary tube to the secondary aliquot. Write the sample ID on the aliquot.

12.Procedure to run Patient sample

• Press Patient order
• Select Assay /Panel, to be run.
• Give Carrier Number and Position number
• Press F3 / Add order
• Put respected carrier in RSH rack

13.Performance Characteristics:

• Linearity: ng/mL
• The limit of detection (LOD): ng/mL
• The limit of quantification(LOQ): ng/mL
• Unit: ng/mL

Normal and critical ranges:

14.Laboratory Clinical interpretation: Specifically, the combined use of serum ferritin levels and mean corpuscular volume (MCV) has made differentiation between iron deficiency, beta-thalassemia trait and normal subjects possible at a very high level of accuracy.

15.Interference and cross reaction: ARCHITECT Ferritin assay demonstrated ≤ 10% mean interference at the levels indicated below.

• Hemoglobin 200 mg/dL
• Bilirubin 20 mg/dL
• Triglycerides 3000 mg/dL
• Protein 2 g/dL and 12 g/dL

16.Potential source of variation: Turn around time (TAT): Routine: 6.0 hours Urgent: 2.0 hours

17.Recording of observation:

• Software backup
• Machine raw data

18.Storage & Disposal of waste: Follow storage & discard procedure

19.Environmental & Safety control: Contact with acids liberates very toxic gas. Dispose of contents / container in accordance with local regulations.

20.References:

1. Krause JR, Stolc V. Serum Ferritin and Bone Marrow Iron Stores
2. Correlation with Absence of Iron in Biopsy Specimens. Am J ClinPathol 1979;72:817-820.
3. Skikne BS, Cook JD. Serum Ferritin in the Evaluation of Iron Status.Lab Management 1981;19:31-35.
4. Addison GM, Beamish MR, Hales CN, et al. An ImmunoradiometricAssay for Ferritin in the Serum of Normal Subjects and Patients withIron Deficiency and Iron Overload. J Clin Pathol 1972;25:326-329.
5. Jacobs A, Miller F, Worwood M, et al. Ferritin in the Serum of NormalSubjects and Patients with Iron Deficiency and Iron Overload. Br MedJ 1972;4:206-208.
6. Lipschitz DA, Cook JD, Finch CA. A Clinical Evaluation of SerumFerritin as an Index of Iron Stores.