2019/04/25

Clinical Chemistry A93A01316CUS

A11A01635

24 mL
ABX Pentra Bilirubin, Direct CP 7 mL

■ Pentra C400

Diagnostic reagent for quantitative in vitro determination of Direct Bilirubin

in serum or plasma by colorimetry.

Application Release delayed function of enzymes for bilirubin degradation.

Common bilirubin methods detect either total bilirubin or
direct bilirubin. Determinations of direct bilirubin measure
Serum, plasma: Bili-D mainly conjugated, water soluble bilirubin. Unconjugated
1.xx bilirubin can therefore be estimated as the difference
between total bilirubin and direct bilirubin.

Intended Use
Method (3)
ABX Pentra Bilirubin, Direct CP reagent is intended for
the quantitative in vitro diagnostic determination of direct Photometric test using 2,4‑dichloroaniline (DCA).
bilirubin in human serum and plasma based on a Direct bilirubin in presence of diazotized
photometric test using 2,4-dichloroaniline (DCA). 2,4‑dichloroaniline forms a red colored azocompound in
Measurements of the levels of bilirubin (direct or total), an acidic solution.
organic compound formed during the normal and
abnormal destruction of red blood cells, are used in the
diagnosis and treatment of liver, hemolytic hematological, Reagents
and metabolic disorders, including hepatitis and gall
bladder block.
ABX Pentra Bilirubin, Direct CP is ready-to-use.

Clinical Interest (1, 2) Reagent 1:

EDTA-Na2 0.1 mmol/L
Bilirubin is a breakdown product of hemoglobin. Free, NaCl 150 mmol/L
unconjugated bilirubin is extremely apolar and nearly
Sulfamic acid 100 mmol/L
insoluble in water, thus forming a complex with albumin
for the transport in the blood from the spleen to the liver.
In the liver, bilirubin is conjugated with glucoronic acid Reagent 2:
and the resulting water soluble bilirubin glucoronides are 2,4-Dichlorophenyl-diazonium salt 0.5 mmol/L
excreted via the bile ducts. HCl 900 mmol/L
Hyperbilirubinemia can be caused by increased bilirubin
EDTA-Na2 0.13 mmol/L
production due to hemolysis (pre-hepatic jaundice), by
parenchymal damages of the liver (intra-hepatic jaundice)
or by occlusion of bile ducts (post-hepatic jaundice). A ABX Pentra Bilirubin, Direct CP should be used
chronic congenital (predominantly unconjugated) according to this notice. The manufacturer cannot
QUAL-QA-WORI-5542 Rev.1

hyperbilirubinemia called Gilbert’s syndrome is quite guarantee its performance if used otherwise.
frequent in the population. High levels of total bilirubin are
observed in 60-70% of neonates due to an increased
postpartal breakdown of erythrocytes and because of

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 Clinical Chemistry

ABX Pentra Bilirubin, Direct CP

Handling a Specimen

1. Remove both caps of the cassette. ■ Serum.

■ Plasma in lithium heparin.
2. If present, remove foam by using a plastic pipette.
3. Position the protective cap (GBM0969) on the Anticoagulants other than those listed have not been
cassette. tested by HORIBA Medical and are therefore not
4. Place the cassette into the refrigerated reagent recommended for use with this assay.
compartment.
Stability (4, 1):

Calibrator ■ At 20-25°C: 2 days

■ At 4-8°C: 7 days
For calibration, use: ■ At -20°C: 6 months (in case of immediate freezing)
ABX Pentra Multical (A11A01652) (not included)
10 x 3 mL (lyophilisate) It is very important to store the sample protected from
light!
In the case of intensive sun irradiation: decrease in total
Control b bilirubin by up to 30% after 1 hour.
Freeze only once.
For internal quality control, use:

■ ABX Pentra N Control / ABX Pentra N MultiControl

Reference Range (1)
(A11A01653 / 1300054414) (not included)
10 x 5 mL (lyophilisate)
■ ABX Pentra P Control / ABX Pentra P MultiControl Each laboratory should establish its own reference
(A11A01654 / 1300054415) (not included) ranges. The values given here are used as guidelines only.
10 x 5 mL (lyophilisate)
Adults and children: ≤ 0.2 mg/dL (≤ 3.4 µmol/L).
Each control should be assayed daily and/or after a
calibration.
The frequency of controls and the confidence intervals Storage and Stability
should correspond to laboratory guidelines and country-
specific directives. You should follow federal, state and
local guidelines for testing quality control materials. The Stability before opening:
results must be within the range of the defined confidence
Stable up to the expiry date on the label if stored at
limits. Each laboratory should establish a procedure to
2-8°C.
follow if the results exceed these confidence limits.

Stability after opening:

Materials Required but not Provided b
Refer to the paragraph "Performance on Pentra C400".

■ Automated clinical chemistry analyzer: Pentra C400 Do not freeze.

■ Calibrator: ABX Pentra Multical (A11A01652)
■ Controls:
ABX Pentra N Control / ABX Pentra N MultiControl Waste Management
(A11A01653 / 1300054414)
ABX Pentra P Control / ABX Pentra P MultiControl
(A11A01654 / 1300054415) Please refer to local legal requirements.
■ Standard laboratory equipment.

aModification: correction of handling.

bModification: new control.

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 Clinical Chemistry

ABX Pentra Bilirubin, Direct CP

General Precautions Mean value Mean value

CV %
µmol/L mg/dL
■ This reagent is for professional in vitro diagnostic use Control
15.33 0.90 0.67
specimen 1
only.
■ For prescription use only. Control
31.64 1.85 0.44
■ This reagent is classified as hazardous in compliance specimen 2
with regulation (EC) N°.1272/2008. Specimen 1 4.01 0.23 3.23
■ Warning Specimen 2 25.92 1.52 0.59
H290: May be corrosive to metals.
Specimen 3 134.63 7.88 2.69
P234: Keep only in original container.
P390: Absorb spillage to prevent material damage.
P406: Store in corrosive resistant container with a Reproducibility (total precision)
resistant inner liner. Reproductibility according to the recommendations found
■ Observe the standard laboratory precautions for use. in the CLSI (NCCLS), EP5-A2 protocol (6) with samples
■ The reagent cassettes are disposable and should be tested in duplicate for 20 days (2 series per day):
disposed of in accordance with the local legal
requirements. ■ 2 controls
■ Please refer to the SDS associated with the reagent. ■ 2 specimens (low / high levels)
■ Do not use the product if there is visible evidence of
biological, chemical or physical deterioration. Mean value Mean value
■ It is the user's responsibility to verify that this CV %
µmol/L mg/dL
document is applicable to the reagent used.
Control
16.02 0.94 4.26
specimen 1
Control
Performance on Pentra C400 specimen 2
34.56 2.02 4.22

Specimen 1 11.72 0.69 3.27

Serum, plasma
Specimen 2 65.40 3.83 2.98

The performance data listed below are representative of

Measuring Range
performance on HORIBA Medical Systems.
The assay confirmed a measuring range from 0.69 µmol/L
Number of tests: 100 tests (0.04 mg/dL) to 116 µmol/L (6.79 mg/dL).
The measuring range is extended up to 580 µmol/L
On Board Reagent Stability (33.9 mg/dL) with the automatic post-dilution.
The reagent linearity has been assessed up to 116 µmol/L
Once opened, the reagent cassette placed in the (6.79 mg/dL) according to the recommendations found in
refrigerated Pentra C400 compartment is stable for 30 the CLSI (NCCLS), EP6-A protocol (7).
days.

Sample volume: 25 µL/test Correlation

Patient samples: Serum
Detection Limit Number of patient samples: 124
Specimens are correlated with a commercial reagent
The detection limit is determined according to the Valtec taken as reference according to the recommendations
protocol (5) and equals 0.69 µmol/L (0.04 mg/dL). found in the CLSI (NCCLS), EP9-A2 protocol (8).
Values ranged from 1.5 µmol/L (0.09 mg/dL) to
Accuracy and Precision 115 µmol/L (6.71 mg/dL).
The equation for the allometric line obtained using
Repeatability (within-run precision) Passing-Bablock regression procedure (9) is:
Y = 1.06 X + 0.7 (µmol/L)
Repeatability according to the recommendations found in Y = 1.06 X + 0.04 (mg/dL)
the Valtec protocol (5) with samples tested 20 times: with a correlation coefficient r2 = 0.9928.
■ 2 controls
■ 3 specimens (low / medium / high levels)

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 Clinical Chemistry

ABX Pentra Bilirubin, Direct CP

Interferences 9. Passing H, Bablock W. A new biometrical procedure

for testing the equality of measurements from two
Haemoglobin: Do not use hemolysed samples.
different analytical methods. J. Clin. Chem. Clin.
Triglycerides: No significant influence is observed up to
Biochem. (1983) 21: 709-20.
an Intralipid® concentration
10. Young DS. Effects of Drugs on Clinical Laboratory
(representative of lipemia) of 7 mmol/L
Tests. 4th Edition, Washington, DC, AACC Press
(612.5 mg/dL).
Other limitations are given by Young as a list of drugs and (1997) 3: 143-163.
preanalytical variables known to affect this methodology 11. Young DS. Effects of Preanalytical Variables on
(10, 11). Clinical Laboratory Tests. 2nd Edition, Washington,
DC, AACC Press (1997) 3: 120-132.
Calibration Stability
The reagent is calibrated on Day 0. The calibration
stability is checked by testing 2 control specimens.
The calibration stability is 10 days.
Note: A recalibration is recommended when reagent lots
change, and when quality control results fall outside the
range established.

Conversion Factor
µmol/L x 0.585 = mg/L
µmol/L x 0.0585 = mg/dL

Reference
1. Thomas L. ed. Clinical Laboratory Diagnostics. 1st
ed. Frankfurt: TH-Books Verlagsgesellschaft (1998):
192-202.
2. Tolman KG, Rej R. Liver function. In: Burtis C.A.,
Ashwood E.R., editors. Tietz Textbook of Clinical
Chemistry. 3rd ed. Philadelphia: WB Saunders
Company (1999): 1125-1177.
3. Rand RN, Di Pasqua A. A new diazo method for the
determination of bilirubin. Clin. Chem. (1962) 6:
570-8.
4. Use of Anticoagulants in diagnostics laboratory
investigations. WHO publication WHO/DIL/LAB/99.1
Rev.2 (2002): 46.
5. Vassault A, Grafmeyer D, Naudin C et al. Protocole
de validation de techniques (document B). Ann. Biol.
Clin. (1986) 44: 686-745.
6. Evaluation of Precision Performance of Quantitative
Measurement Method. Approved Guideline, CLSI
(NCCLS) document EP5-A2 (2004) 24 (25).
7. Evaluation of the Linearity of Quantitative Analytical
Methods. Approved Guideline, CLSI (NCCLS)
document EP6-A (2003) 23 (16).
8. Method Comparison and Bias Estimation Using
Patient Samples. Approved Guideline, 2nd ed., CLSI
(NCCLS) document EP9-A2 (2002) 22 (19).

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