UMA CO., LTD.

MEASURE D-Bil
2-19-6 Yokosuka Reagent for measuring Direct Bilirubin
Matsudo, Chiba, Japan Enzymatic

á 2 ~ 8 °C IVD In vitro Diagnostics Packages

R1 1 ´ 90 mL R2 1 ´ 30 mL
T DO NOT freeze 6 18 months/block from light R1 1 ´ 60 mL R2 1 ´ 20 mL

1. PURPOSE OF USE 6. MEASUREMENT PRINCIPLE

In vitro determination of Direct Bilirubin in blood plasma
and blood serum.
2. GENERAL INSTRUCTION
1. For in vitro diagnostics use only.
2. Diagnosis should be made in a comprehensive manner,
in accordance with other related test results and clinical
Direct bilirubin in serum is oxidized to biliverdin by the
action of bilirubin oxidase (BOD) in citric acid - lactic acid
buffer (pH 3.5 ~ 3.8), at this point bilirubin-dependent
absorbance of 450 nm will decrease. Direct bilirubin
concentration can be obtained from decreased amount of
absorbance caused by this reaction.

symptoms by the doctor in attendance. 7. STANDARD MEASUREMENT OPERATION

3. For guaranteed results, usage of this product must Specimen Calibrator Blank
comply with the instruction in this manual. (S) (Std) (B)
4. For automatic analyzers: follow their instructions. Specimen (µL) 20 - -

3. MATERIALS REQUIRED BUT NOT INCLUDED Calibrator (µL) - 20 -

- Saline 0.9 % and high grade purified water Saline (µL) - - 20

- Micropipet and other basic laboratory equipment. R-1 (µL) 540 540 540

- Calibrators and Controls (separatedly sold) Incubate at 37 °C in 5 minutes

R-2 (µL) 180 180 180
4. REAGENT COMPOSITION & PREPARATION
Mix well; incubate at 37 °C for 5 minutes; measure
- Reagent R-1: Citric acid - lactic acid buffer
absorbance at 450/600 nm
Reagent R-1 is ready for use
Note: See sample preparation for details of specimen
- Reagent R-2: Bilirubin Oxidase (BOD)
Reagent R-2 is ready for use 8. CALCULATION & UNIT CONVERSION
- Calibrator (separatedly sold): Ready for use Calculation
- Controls Lyo-1 & Lyo-2 (separatedly sold): Put 1 mL of - Calculate ∆Abs of specimen & standards vs blank
purified water to the vials of controls (L, H); leave at room - Plot a calibration curve D-Bil (mg/dL) = f(∆Abs)
temparature for 30 minutes before use. After reconstituted, - Calculate D-Bil concentration in specimen using the
controls can be use without dilution. curve (doing same procedure for Controls)

5. SAMPLE PREPARATION & STORAGE Unit conversion

- Serum: Wait until blood sample completly coagulated. 1 mg/dL = 17.1 μmol/L

Take the supernatant to use as specimen. 9. PERFORMANCE & CORRELATION TEST

- Plasma: Treat blood sample by anticoagulant; leave it to
stand for 3 hours or centrifuge at 2000 rpm for 2 minutes;
take the plasma layer (supernatant) and use as specimen.
- Analyze sample soon after collection. In case, it could
not be analyzed soon, store sample 2 ~ 8 °C and analyze
within 3 days.
- Avoid to use hemolytic sample
- Bilirubin is sensitive to light, avoid expose sample to light
after collection.
Performance
- Sensitivity: When measuring purified water, absorbance
ranges from 0.00 ~ 0.05. When measuring the solution of 9
mg/dL direct bilirubin as sample, change in absorbance
ranges from -0.270 ~ -0.220 absorbance unit.
- Specificity: The accuracy is within ±20.0%.
- Reproducibility: CV value < 5.0%.
- Measuring range: < 25 mg/dL.

1/2 Revision 08/2013

10. EXPECTED VALUES the manual for medical waste provided in each facility.
Normal reference range ・ Sterilize with an autoclave, subjecting them to high
- Less than 0.2 mg/dL. pressure saturated steam at 121 °C for more than 20
Reference range should be established at each facility and minutes. Do not process waste containing sodium
judgement should base on measurement results in a hypochlorite solution with an autoclave.
comprehensive manner together with clinical symptoms ・Immerse at least one hour in sodium hypochlorite solution
and other measurement results. (active chloride concentration of over 1000 ppm).
2. This reagent contains sodium azide. Sodium azide can
11. INTERFERENCES
- There is no influence of hemolysis (hemoglobin) up to 200 react with lead pipe and/or steel pipe and can generate
explosive metal azide. Make sure to use plenty of water at
mg/dL, lipemia of 2,500 formazin turbidity and ascorbic
disposal. Concentration of sodium azide in R-2 is 0.05%.
acid up to 50 mg/dL..
14. OTHER INSTRUCTIONS AND CAUTION
12. INFORMATION FOR AUTOANALYZERS
- Results may differ depending on the sample/reagent
Calculation Method 2-point end (fixed)
ratio. Adjust parameters for different analyzer.
Temperature 37 °C
- Prepare the calibration curve on the day of
Specimen 5 determination.
Volume (μL) R1 135
R2 45
Main 450
Wavelength (nm)
Sub- 600
Point 1 10
Measurement
Point 2 16
(cycle) Point 3 34

Calibration type Linear

Unit mg/dL

13. HANDLING, USAGE & DISPOSAL

Handling
1. Specimen can be potentially positive for infectious agents
including hepatitis B virus and HIV. Wear glove and goggle
when needed.2. In case reagents got into skin, eye or
mouth by mistake, wash it immediately with plenty of water
and consult the doctor if needed.
3. If reagents are spilled, dilute with water and wipe it out. If
specimen is spilled, spray 80% of alcohol over the
specimen and wipe it out.
Usage
1. Store reagents under specified condition. Do not use
after expiration date.
2. Do not use the container and auxiliaries included in this
kit for other purposes.
3. Do not mix reagents of different lot for use.
4. Do not add to the reagent being used even if it is the
same lot number.
Disposal
1. All specimens, as well as all instruments (e.g. test tubes)
that come in contact with the specimens, must be treated by
the following methods, or they must be treated according to

2/2 Revision 08/2013