UIBC Examination Procedure

1.Purpose of examination: UIBC estimation from serum or plasma by Ferene 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: R1: Glycyl glycine buffer (pH 8.6) 255 mmol/L

• Sodium bicarbonate 46 mmol/L
• Iron (II) chloride 20 μmol/L

R2:Ascorbic acid (pH 2.5) 300 mmol/L

• Ferene-S 12.5 mmol/L

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
• Reagent Storage and stability
• Unopened reagent stable at 2-8°C until expiration date.
• On board System temperature reagent is stable for 28 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.

7.Calibration Procedure:

• UIBC Calibrator
• 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 aliquot.
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.

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

Procedure for Reconstitution of IQC

1. Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
2. Leave to stand for 30 min in the dark place.
3. Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
4. 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.
5. Prepare aliquots of 150 µl from the reconstituted QC material.
6. Store these aliquots at -15° C to -20° C.
7. 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.

9.Principle of the procedure used for examinations:

• Sample is added to an alkaline buffer containing a known concentration of iron to saturate the available binding sites on transferrin. The iron that remains free after transferrin saturation is reduced to a ferrous state and then complexed by Ferene-S* to form a stable complex, of which the color intensity is measured at 604 nm. The color intensity is directly proportional to the unbound excess iron concentration and indirectly proportional to the unsaturated iron-binding capacity. UIBC is therefore determined by subtracting the quantity of unbound iron from the total added quantity.

10.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.

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

11.Performance Characteristics:

1. Linearity: 25 to 500 μg/dL
2. The limit of quantitation (LOQ) : ≤ 25 μg/dL
3. Unit: μg/dL

Normal and critical ranges:

12.Laboratory Clinical interpretation: UIBC and TIBC are increased in low iron states such as uncomplicated anemia and decreased in high iron conditions such as hemochromatosis. The lone exception to the preceding is the case of anemia of chronic disease where the patient may be anemic but has adequate iron reserves and a low UIBC.

13.Interference and cross reaction:

• No interference from Bilirubin up to 6.6 mg/dL
• No interference from Intralipid up to 600 mg/dL
• No interference from Sulfapyrdine up to 300mg/dl
• No interference from Temozolomyde up to 20 mg/dl

14.Potential source of variation: Turn around time (TAT):

• Routine: 6.0 hours
• Urgent: 2.0 hours

15.Recording of observation:

• Software backup
• Machine raw data

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

17.Environmental & Safety control:

Prevention

• void breathing mist / vapors / spray.
• Contaminated work clothing should not be
• allowed out of the workplace.
• Wear protective gloves / protective
• clothing / eye protection.

Response:

• IF ON SKIN: Wash with plenty of water.
• If skin irritation or rash occurs: Get medical advice / attention.
• Take off contaminated clothing and wash it before reuse.

Disposal :

• Dispose of contents / container in accordance with local regulations.

18.References:

1. US department of labor,Occupational safety and health administration.
2. US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories.
3. World health organization. Biosafety manual,3rd edition
4. ARCHITECT CK MB 6K25-30 307233/R0