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                      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:** 
  - Type of Sample: Serum,  Plasma
  - Type of container and additives: Plain without any additives 
  - Patient Preparation: As per Primary Sample Collection Manual [[sample_collection_manual|]]
  - Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
  - Handling and transport: As per Primary Sample collection manual 
  - 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:
  - Reagent lot change
  - QC out of range
  - After service or maintenance
  - Replacement in any parts of Instrument

**Procedure:**
  - Start the equipment.WDI abbotte fully.docx
  - Calibrators are ready to use.
  - Put calibrator 15-20 minutes at room temperature  
  - Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation. 
  - Take a 150 µl calibrator solution in to separate aliquots.
  - Go to the calibration and give the calibration order.
  - Verify calibration with at least two levels of controls
  - 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
  - Press Control order 
  - Select Assay /Panel, to be run.
  - Select the control/s and its level/s
  - Give Carrier Number and Position number
  - Press F3 / Add order
  - Put respected carrier in RSH rack
  - Check IQC results, in case outliers call residents.

**10.Principle of the procedure used for examinations:**
  - Sample, and anti-ferritin coated paramagnetic microparticles are combined. The ferritin present in the sample binds to the anti- ferritin coated microparticles.
  - After washing, anti-ferritin acridinium-labeled conjugate is added to create a reaction mixture.
  - Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction mixture.
  - 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:**

^1 mo^200-600^ng/mL^
^0-1 Y^50-200^ng/mL^
^1-15 y^7-140^ng/mL^
^Male^20-250^ng/mL^
^Female^10-120^ng/mL^

**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:**
  - Krause JR, Stolc V. Serum Ferritin and Bone Marrow Iron Stores 
  - Correlation with Absence of Iron in Biopsy Specimens. Am J ClinPathol 1979;72:817-820.
  - Skikne BS, Cook JD. Serum Ferritin in the Evaluation of Iron Status.Lab Management 1981;19:31-35.
  - 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.
  - 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.
  - Lipschitz DA, Cook JD, Finch CA. A Clinical Evaluation of SerumFerritin as an Index of Iron Stores.

^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^
^Document No.1^**Document Name**: Ferritin Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^
^Issue No. : 01^Issue Date :30/04/2024^Page No.^^
^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^