STANDARD OPERATING PROCEDURE

QUANTITATIVE IN VITRO DETERMINATION OF BILIRUBIN TOTAL

IN HUMAN SERUM

LOCATION Chandigarh COPY NO. 1

SOP NAME Quantitative in vitro determination of Bilirubin total in human serum

SOP NO. BI/EM 200/SOP-05/V-I

DEPARTMENT Cl. Biochemistry SUPERSEDES None

Signature & Date Signature & Date

PREPARED BY REVIEWED BY
Sr. Technician Lab Director
28-09-2020
26-09-2020

APPROVED BY ISSUED BY
Lab Director Quality Manager
28-09-2020 30-09-2020

EFFECTIVE DATE 05-10-2020

Purpose of the examination:

To establish the standard operating procedure for quantitative in vitro determination of Bilirubin total in
human serum.

Principle and method of the procedure used for examination:

Walter & Gerarde Method

In the determination of Bilirubin Total, Bilirubin is coupled with diazotized sulfanilic acid in the presence
of ethylene glycol and dimethylsulfoxide as solvents to produce an intensely colored diazo dye. The
intensity of color of this solution is proportional to the concentration of the bilirubin total in the sample

Responsibility:
Primary Responsibility: All scientific officers involved in analysis of samples for this test shall follow
the SOP.
Secondary Responsibility: It is the responsibility of the Section head/Deputy Section Head to ensure
implementation of the same. In case any deviation is planned or observed it should be notified to Quality
Manager/ Technical Supervisor and Lab Director immediately.

Abbreviations & Definitions:

LIST OF ABBREVIATIONS
% Percentage SD Standard deviation
CV coefficient of variation °C Degree Celsius
CAL Calibrator AMR Analytical Measurement Range
g/dL Gram per deci litre NA Not applicable
QC Quality Control

Performance Characteristics:
• Limit of quantification: 0.08 mg/dl

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BI/EM 200/SOP-05/V-I
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 STANDARD OPERATING PROCEDURE
QUANTITATIVE IN VITRO DETERMINATION OF BILIRUBIN TOTAL
IN HUMAN SERUM

• Linearty: 23 mg/dl
• Measuring range: 0.08 – 23 mg/dl
• Precision
Intra-assay precision Within run (n = 20) Mean (mg/dl) SD (mg/dl) CV (%)

Sample – 1 1.58 0.015 0.93

Sample – 2 3.90 0.035 0.90
Inter-assay precision Run to run (n=20) Mean (mg/dl) SD (mg/dl) CV (%)

Sample – 1 1.36 0.023 1.67

Sample – 2 5.23 0.068 1.31

Type of Sample: Serum

Patient Preparation:
Use unheamolytic serum or plasma (heparin, EDTA).
It is recommended to follow NCCLS procedures (or similar standardized conditions).
Stability: 2 days at 15–25°C
7 days at 2–8°C
3 months at -20°C
FREEZE ONLY ONCE!
Discard contaminated specimens.

Type of Container and additives: SST or Heparin vacutainer. No additive is needed

Required Equipment and Reagent:

• Equipment: EM 200 analyzer.
• Active Ingredients Concentration
R1
Sulphanilic Acid 28.87 mmol/l
HCl 58.8 mmol/l
Cetrimide 68.6 mmol/l
R2
Sodium Nitrite 2.9 mmol/l

Reagent Preparation
Reagent is liquid, ready to use.

Reagent Storage:
The unopened reagents are stable till the expiry date stated on the bottle and kit label when stored at
2–8°C. Before opening let R1 equilibrate for 10 minutes at room temperature.
On board stability: min. 30 days if refrigerated (2–8°C) and not contaminated.

Indications of Reagent Deterioration

Turbidity.
Failure to recover control values within the assigned range.

Environments and safety controls:

Shoud be handled by entitled and professionally trained person. Reagent is classified as eye and skin
irritant. It contains 2.5% cetrimide and 0.5% sulphanilic acid. It may cause an allergic skin reaction

Required Equipment and Reagent:

Equipment: EM 200 analyzer.
Active Ingredients Concentration
R1
Sulphanilic Acid 28.87 mmol/l
HCl 23 mmol/l
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BI/EM 200/SOP-05/V-I
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 STANDARD OPERATING PROCEDURE
QUANTITATIVE IN VITRO DETERMINATION OF BILIRUBIN TOTAL
IN HUMAN SERUM

R2
Sodium Nitrite 2.9 mmol/l

Reagent Preparation
Reagent is liquid, ready to use.
Reagent Storage:
The unopened reagents are stable till the expiry date stated on the bottle and kit label when
stored at 2–8°C.
On board stability: min. 30 days if refrigerated (2–8°C) and not contaminated.

Indications of Reagent Deterioration

Turbidity.
Failure to recover control values within the assigned range.

Environments and safety controls:

Should be handled by entitled and professionally trained person. Reagent of the kit are not classified
like dangerous reagent 1 containes a small amount of sulfanilic acid and may cause an allergic skin
reaction. Reagent 2 containes less than 0.02% sodium nitrite classified as toxic dangerous substance
for the environment.

Calibration Procedure:
Calibration with serum based XL Multical calibrator is recommended.
Calibration frequency: it is recommended to do a calibration
• After reagent lot change
• As required by internal quality control procedures

Procedural Steps:
Refer SOP BI/EM 200/SOP-01 Operating Procedure of EM 200
• Assay Time: 1 hr
• Dilution or concentration, if required: NA

Quality Control (QC) Procedure:

Refer SOP BI/EM 200/SOP-01 Operating Procedure of EM 200 for procedure of running QC.
Run minimum two level QC daily or refer IQC plan.
Refer MSP-28 Internal Quality Control for review of QC values

Interferences cross reactions and potential sources of variation:

Following substances do not interfere:
Haemoglobin up to 7.5 g/l,
Triglycerides up to 1500 mg/dl

Result calculation & Interpretation:

Results are calculated automatically by the instrument.

Biological Reference Interval:

Adults: 0 – 2.0 mg/dl
Cord < 2 mg/dl
Newborns, premature:
0 – 1 d 1.0 – 8.0 mg/dl
1 – 2 d 6.0 – 12.0 mg/dl
3 – 5 d 10.0 – 14.0 mg/dl
Newborns, full term:
0 – 1 d 2.0 – 6.0 mg/dl
1 – 2 d 6.0 – 10.0 mg/dl
3 – 5 d 4.0 – 8.0 mg/dl

Measurement Uncertainty: Defined as 2 x CV%

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 STANDARD OPERATING PROCEDURE
QUANTITATIVE IN VITRO DETERMINATION OF BILIRUBIN TOTAL
IN HUMAN SERUM

Reportable interval of examination results: 0.08 – 23 mg/dl

Alert /Critical Value: > 15 mg/dL

Laboratory clinical interpretation:

Bilirubin is a breakdown product of haemoglobin. Bilirubin formed in the reticulo endothelial system is
transported bound by albumin to the liver. This bilirubin is water insoluble and is known as indirect or
unconjugated bilirubin. In the liver, bilirubin is conjugated to glucuronic acid to form direct bilirubin.
Conjugated bilirubin is excreted via the biliary system into the intestine. Here it is metabolised by
bacteria to urobilinogen & stercobilinogen.

TOTAL BILIRUBIN = INDIRECT BILIRUBIN + DIRECT BILIRUBIN

References:
1. Operating manual EM200
2. Kit insert

Review Log
Signature Date Remark

REVIEWED ON & BY
(Technical Supervisor/
Lab Director)

Revision Log
Version No Effective Date Revision Description

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