Direct Bilirubin Examination Procedure

1.Purpose of examination: Bilirubin Direct estimation from serum or plasma by Diazo end point Method.

2.Responsibility and Authority:

1. Calibration: Technician
2. Quality Control: Technician
3. Routine operation: Technician
4. 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. Sulphanilic acid 29.0 mmol/L
• Hydrochloric acid 0.17 mmol/L
• R2. Sodium nitrite 38.5 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:

• Billi Calibrators- Level 1 & 2
• Frequency:

1. Reagent lot change
2. QC out of range
3. After service or maintenance
4. 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 both level of calibrator solution in to separate aliquot.
• 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.

8.Quality control Procedure:

• Name: Biorad Level 2 & 3
• 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: Billirubin reacts with diazotized sulphanilic acid to form a coloured azobillirubin compound.The increase in absorbance at 540 nm due to azobillirubin is proportional to the direct billirubin concentration.

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.

11.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

12.Performance Characteristics:

• Linearity: 0.1 to 15.0 mg/dL

If values exceed this linearity limit 15.00 mg/dl, dilute the sample by Manual Dilution Procedure, or the Automatic Dilution Protocol provided in the assay parameters.

Automated Dilution Protocol: When using the Automated Dilution Protocol, the system performs a dilution of the specimen and automatically corrects the concentration by multiplying the result by the appropriate dilution factor.

Manual Dilution Procedure: Dilute the specimen with saline (0.85% to 0.90% NaCl). Enter the dilution factor in the Patient or Control order screen

• The limit of detection (LOD): 0.04 mg/dl
• The limit of quantification(LOQ): ≤ 0.1 mg/dL
• Unit: mg/dl

Normal and critical ranges: For Newborn:

Adults and infants: 0-2.0 mg/dL

13.Laboratory Clinical interpretation:

• Unconjugated hyper-bilirubinemia is seen in newborns, in increased red cell destruction (hemolytic anemia, extension hematoma), is ineffective erythropoiesis some rare genetic disease (Gilbert's syndrome, Crigler- Najjar syndrome)
• Conjugated hyper-bilirubinemiais associated to a decreased excretion of bile due to liver disease (hepatitis or cirrhosis) or to intrahepatic or extrahepatic cholestasis

14.Interference and cross reaction: The Following analytes were tested up to the levels indicated at Direct Bilirubin concentrations of 0.14mg/dl and 5.03 mg/dl, and found not to interfere:

• No interference from Hemoglobin up to 31 mg/dL
• No interference from Intralipid up to 125 mg/dL
• No interference from Human triglyceride up to 519 mg/dl
• No interference from Indocynine green up to 6.3 mg/dl

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

• Routine: 6.0 hours
• Urgent: 2.0 hours

16.Recording of observation:

• Software backup
• Machine raw data

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

18.Environmental & Safety control: Reagent R1 contains sufamic acid that Causes severe skin burns and eye Damage .

19.Precautions:

1. Wear protective gloves / protective clothing / eye protection
2. Do not breathe mist / vapors / spray
3. Wash hands thoroughly after handling
4. Keep only in original container

20.Response

• IF SWALLOWED: Rinse mouth. Do NOT induce vomiting.
• IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.
• IF IN EYES: Rinse cautiously with water for several minutes.Remove contact lenses, if present and easy to do. Continue rinsing.Absorb spillage to prevent material damage.

21.References:

1. Jacobs DS, DeMott WR, Grady HJ, et al. Laboratory Test Handbook, 4th ed. Hudson, OH: Lexi‑Comp; 1996:86.
2. Dennery PA, Seidman DS, Stevenson DK. Drug therapy: neonatal hyperbilirubinemia. N Engl J Med 2001;344(8):581–90.
3. Malloy HT, Evelyn KA. The determination of bilirubin with the photoelectric colorimeter. J Biol Chem 1973;119:481–90.
4. Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry, 3rd ed. Philadelphia, PA: WB Saunders; 1999:1136–7.
5. Walters MI, Gerarde HW. An ultramicromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchemical J 1970;15:231–43.
6. ARCHITECT TOTAL BILI 6L45 307147/R04
7. NABL 112 effective from 01/06/2019