direct_billirubin [Laboratory Service Sir T Hospital , Bhavnagar ]

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                      Direct Bilirubin Examination Procedure
**1.Purpose of examination:** 
Bilirubin Direct estimation from serum or plasma by Diazo end point 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:**
  * 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:
  - 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 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** 
  - 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.

**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:**
^Cord( Premature) ^<2.0 mg/dL^
^Cord( full term))^ <2.0 mg/dL^
^0-1 days (Premature) ^1-8 mg/dL^
^0-1 days (Full term) ^ 2-6 mg/dL^
^1-2 days (Premature)^ 6-12 mg/dL^
^1-2 days (Full term) ^6-10 mg/dL^
^3-5 days (Premature) ^ 10.0-14.0 mg/dL^
^3-5 days (Full term) ^4.0-8.0 mg/dL^

**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:**
  - Wear protective gloves / protective clothing / eye protection
  - Do not breathe mist / vapors / spray
  - Wash hands thoroughly after handling
  - 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:**
  - Jacobs DS, DeMott WR, Grady HJ, et al. Laboratory Test Handbook, 4th ed. Hudson, OH: Lexi‑Comp; 1996:86.
  - Dennery PA, Seidman DS, Stevenson DK. Drug therapy: neonatal hyperbilirubinemia. N Engl J Med 2001;344(8):581–90.
  - Malloy HT, Evelyn KA. The determination of bilirubin with the photoelectric colorimeter. J Biol Chem 1973;119:481–90.
  - Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry, 3rd ed. Philadelphia, PA: WB Saunders; 1999:1136–7.
  - Walters MI, Gerarde HW. An ultramicromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchemical J 1970;15:231–43.
  - ARCHITECT  TOTAL BILI 6L45 307147/R04
  - NABL 112 effective from 01/06/2019

|**Printed copy of this document is considered uncontrolled.** It should be compared with controlled electronic copy before use|

^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^
^Document No.1^**Document Name**: Direct Bilirubin 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^