BILIRUBIN

Total & Direct (DCA)

INTENDED USE METHOD
Vitro bilirubin reagent is intended for the in vitro quantitative Diazo method Using dichloroaniline (DCA)
determination of total and direct bilirubin in serum or plasma
on manual system.
CLINICAL SIGNIFICANCE
Bilirubin is formed in the reticuloendothelial system during the degradation of aged erythrocytes. The heme portion from hemoglobin and from other-
containing proteins is removed, metabolized to bilirubin, and transported as a complex with serum albumin to the liver. This process accounts for about
80% of bilirubin formed daily. Other sources of bilirubin include the breakdown of myoglobin and cytochromes and the catabolism of immature red cells
in the bone marrow. In the liver, bilirubin is conjugated with glucuronic acid for solubilization to form conjugated or direct bilirubin for subsequent transport
through the bile duct into the digestive tract where it is metabolized by bacteria to a group of products collectively known as stercobilinogen. Total
bilirubin is the sum of the conjugated and unconjugated fractions. Pre-hepatic diseases or conditions such as hemolytic disease or liver diseases resulting
in impaired entry, transport or conjugation within the liver cause elevation of unconjugated (indirect) bilirubin. Monitoring of bilirubin in newborns,
particularly if premature, has special importance since the hepatic handling of bilirubin is immature leading to elevated unconjugated bilirubin. If not
bound to albumin, unconjugated bilirubin is able to cross the blood brain barrier more easily, increasing the risk of cerebral damage. Total bilirubin is
elevated in conditions causing obstruction of the bile duct, hepatitis, cirrhosis, in hemolytic disorders and several inherited enzyme deficiencies. There
is information indicating elevated levels of direct bilirubin in patients with liver or biliary tract disease, even though, total bilirubin levels are normal.
Therefore, the greatest diagnostic value of direct bilirubin assays stem from their ability to indicate occult liver disease 4.
ASSAY PRINCIPLE
Bilirubin present in the sample reacts with diazotized Dichloroaniline to form a coloured azocomplex whose intensity at 546 nm (540-560 nm) is directly
proportional to the analyte concentration. Detergents present in the reagent for total bilirubin allow to non conjugated bilirubin to solubilize and take part
in the reaction.

EXPECTED VALUES PROCEDURE

Total Bilirubin
Total Bilirubin2 Method 1:
Adults and 0.2 – 1.0 mg/dl
infants > 1 month 3.4 - 17 mol/l Wavelength 546 nm
Newborns premature 10 - 14 mg/dl Cuvette 1 cm light path
Temperature 37 0C
(3-5 days) 171 - 239 mol/l
Zero adjustment against specimen blank
Newborns (3-5 days) 4.0 – 8.0 mg/dl Specimen Serum
68 - 137 mol/l Specimen blank Specimen
Newborns (<48 hrs) 6.0 – 10.0 mg/dl
103 - 171 mol/l R1 1 ml 1 ml
Newborns (<24 hrs) 2.0 – 6.0 mg/dl R2 …… One drop
34 - 103 mol/l Specimen 100 l 100 l
Direct Bilirubin Mix well, let stand 5 minutes at 37oC
Adults and infants Up to 0.2 mg/dl
Up to 3.4 mol/l Method 2: Icetric or pediatric specimen
Specimen blank Specimen
Each laboratory should investigate the transferability of the expected R1 1 ml 1 ml
values to its own patient population and if necessary determine its own R2 …… One drop
reference range. For diagnostic purposes, the bilirubin results should
Specimen 20 l 20 l
always be assessed in conjunction with the patient’s medical history,
clinical examination, and other findings. Mix well, let stand 5 minutes at 37oC

REAGENTS Measure the absorbance of specimen (Aspecimen) against specimen blank.

R1 Total Reagent 2,4 Dichloroaniline 2.7 mmol/l The color is stable for 60 minutes.
HCl 0.29 N
Detergent 30 g/l Direct Bilirubin
Method 1:
R1 Direct Reagent 2,4 Dichloroaniline 2 mmol/l
HCl 0.20 N Wavelength 546 nm
Cuvette 1 cm light path
R2 Sodium nitrite 30.0 mmol/l Temperature 37 0C
• Reagent Preparation & Stability Zero adjustment against specimen blank
All reagents are stable up to the expiry date given on label when stored Specimen Serum
at 2-8 oC. Specimen blank Specimen
R1 1 ml 1 ml
SPECIMEN …… One drop
R2
Serum.
Specimen 100 l 100 l
For direct bilirubin plasma specimen may be used, but the only Mix well, let stand 1 minutes at 370C ( Maximum 2 minutes)
accepted anticoagulants are heparin and oxalate.
• Specimen Preparation & Stability Method 2: Icetric or pediatric specimen
The specimen of choice is serum. Specimens should be assayed Specimen blank Specimen
promptly after collection, since direct bilirubin is reportedly unstable 5. If R1 1 ml 1 ml
testing is delayed, specimens should be protected from exposure to
R2 …… One drop
light. Bilirubin remains stable in serum samples for 2 days at room
temperature, 4 days at 40C, or 3 months at –200C, if care is taken to Specimen 20 l 20 l
prevent exposure to light6. Mix well, let stand 1 minutes at 370C ( Maximum 2 minutes)
Measure the absorbance of specimen (Aspecimen) against specimen blank.
After 2 minutes indirect bilirubin reacts slowly with diazotized
dichloroanilin and leads to over-estimated values.

Vitro Scient, Industrial Area, Basatin Al Ismailia, Belbis, Alsharkia, Egypt. EC REP Medical Device Safety Services
Tel. +20552642664; Email: info@[Link]; Website: [Link] MDSS GmbH, Burckhardtstr. 1
30163 Hannover, Germany.
 CALCULATION

Calculate the bilirubin concentration by using the following formulae: Method Comparison
Comparison studies were carried out using a similar commercially available
For method 1 Bilirubin reagent as a reference. Serum samples were assayed in parallel and
Total Bilirubin Concentration = Specimen absorbance X 15.47 =mg/dl the results compared by least squares regression. The following statistics
were obtained.
Direct Bilirubin Concentration = Specimen absorbance X 15.47 =mg/dl Number of sample pairs 929
Range of sample results 0.05-26 mg/dl
Mean of reference results 1.2 mg/dl
For method 2
Mean of Bilirubin results 1.2 mg/dl
Total Bilirubin Concentration = Specimen absorbance X 71.96 =mg/dl Slope 1.10
Intercept 0.12 mg/dl
Direct Bilirubin Concentration = Specimen absorbance X 71.96 =mg/dl Correlation coefficient 1.00
Sensitivity
Unit conversion The sensitivity is defined as the change of analytical response (∆A/min) per
mg/dl x 17.1= mol/dl unit change in analyte concentration at a pathlength of 1 cm.
QUALITY CONTROL When run as recommended the sensitivity of this assay is 0.01 mg/dl (0.17
It is recommended that controls (normal and abnormal) be included in: mol/l).
• Each set of assays, or LINEARITY
• At least once a shift, or When run as recommended, the assay is linear
• When a new bottle of reagent is used, or up to 20 mg/dl (0.342 mmol/l) for method 1
• After preventive maintenance is performed or a clinical component is up to 100 mg/dl (1.71 mmol/l) for method 2
replaced. If result exceeds 20 mg/dl (0.342 mmol/l) reassay using method 2.
Commercially available control material with established bilirubin values may
be routinely used for quality control. BIBLIOGRAPHY
Failure to obtain the proper range of values in the assay of control material may 1. Jendrassik, L and Grof, P (1938): Biochem. Z. 297: 81.
indicate: 2. Balistreri, WF & Shaw, LM (1987): Liver [Link] Tietz NW, ed.
• Reagent deterioration, Fundamentals of clinical chemistry. 3rd ed. Philadelphia: WB Saunders: 729-
• Instrument malfunction, or
761.
• Procedure errors. 3. Tietz, NW. (1976): Fundamentals of clinical chemistry. W.B. saunders
The following corrective actions are recommended in such situations: Co., Philadelphia, p.1028.
• Repeat the same controls. 4. Gambino, SR et al., (1967): JAMA 201: 1047.
• If repeated control results are outside the limits, prepare fresh control 5. Gordon, ER (1975): The conjugates of bilirubin. Hepatology (NY) 2: 19.
serum and repeat the test. 6. Winkelmann, J, Cannon, DC and Jacobs, SL (1974): Liver function
• If results on fresh control material still remain outside the limits, then tests, including bile pigments. In: Henry RJ, Cannon DC, Winkelman,
repeat the test with fresh reagent. JW, eds. Clinical chemistry, principles and technics. 2 nd ed. New York:
• If results are still out of control, contact Vitro Technical Services.
Harper & Row; 1o42-1079.
7. Young, DS (1990): Effects of Drugs on Clinical Laboratory Tests. Third
INTERFERING SUBSTANCES
• Anticoagulants: Edition. 1990: 3: 6-12.
The only accepted anticoagulants are heparin and oxalate for direct SYMBOL DECLARATION
bilirubin.
• Drugs: Manufacturer
Young7 in 1990 has published a comprehensive list of drugs and
substances, which may interfere with this assay. Consult instructions for use
• Hemolysis:
Avoid hemolyzed specimens. Even slight hemolysis interferes with the test. Batch code (Lot #)
Haemoglobin interference is dependent on both analyte and hemoglobin
Catalog number
concentration.
The interference becomes decreasingly significant with increasing Temperature limitation
concentration of total bilirubin.
• Lipemia: In vitro diagnostic medical device
Avoid lipemic specimens. Even slight lipemia interferes with the test.
Use by
WARNING & PRECAUTION
• Vitro bilirubin reagent is for in vitro diagnostic use only. Normal precautions Caution. Consult instructions
exercised in handling laboratory reagents should be followed.
• The reagent and sample volumes may be altered proportionally to accommodate Keep away from light
different spectrophotometer requirements.
• Valid results depend on an accurately calibrated instrument, timing, and
temperature control. ORDERING INFORMATION
• All specimens must be protected from light. Serum bilirubin will decrease 50%
Product REF SIZE
in one hour if kept at room temperature and at direct sunlight.
• Don’t use the reagent if it is turbid.
Bilirubin T&D 18201 2 x 100 ml
PERFORMANCE CHARACTERISTICS
Imprecision
Reproducibility was determined using in an internal protocol. The following
results were obtained.
Within Run Between Day Article # :182 -EN
I. Total Bilirubin Date of Revision :03/2021
Control Level I Level II Control Level I Level II
Number of samples 40 40 Number of samples 40 40
Mean (mg/dl) 1.15 4.22 Mean (mg/dl) 1.15 4.27
SD (mg/dl) 0.02 0.04 SD (mg/dl) 0.02 0.13
CV (%) 2.02 1.05 CV (%) 1.91 3.2
II. Direct Bilirubin
Number of samples 40 40 Number of samples 40 40
Mean (mg/dl) 0.78 2.28 Mean (mg/dl) 0.8 2.18
SD (mg/dl) 0.015 0.01 SD (mg/dl) 0.01 0.03
CV (%) 1.31 0.65 CV (%) 1.63 1.53
ISO
13485:2016

Vitro Scient, Industrial Area, Basatin Al Ismailia, Belbis, Alsharkia, Egypt. EC REP Medical Device Safety Services
Tel. +20552642664; Email: info@[Link]; Website: [Link] MDSS GmbH, Burckhardtstr. 1
30163 Hannover, Germany.