1. AEROSET & ARCHITECT cSystems
Total Bilirubin (BiliT) 6L45
Revised Training Presentation
Reduced sample volume (2.6 µL) March 2010
REF
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2. * Background: BiliT sample volume reduction
In May 2009, a complaint for the reduced recovery of bilirubin in a neonatal sample was received. Investigation showed that increased concentrations of unconjugated bilirubin can result in reduced bilirubin recovery.
Customers were notified via FA09JUL2009 and instructed to dilute samples with a bilirubin concentration greater than 15 mg/dL (257 µmol/L. Sample dilution was demonstrated to provide correct results.
A Level-III investigation was opened to investigate the under-recovery at higher concentrations. The results concluded that a reduction in sample volume to 2.6 µL would resolve the under-recovery issue. Verification testing confirmed that acceptable assay performance was achieved with a sample volume of 2.6 µL.
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C8000 PN v.1

3. This training document has been revised to include information, where necessary, that is specific for the 2.6 µL sample volume. Obsolete information has been deleted.
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4. BiliT Agenda Clinical Utility Methodology Sample Handling
Reagent Handling
Calibration
Reportable Range
Interference
Precision
Method Comparison
Assay-specific Information
Troubleshooting Tips
Questions and Answers
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5. Bilirubin Clinical Utility
Bilirubin Synthesis
Old RBCs broken down by spleen
Heme converted to unconjugated bilirubin
Unconjugated bilirubin transported to liver bound to albumin
Bilirubin is conjugated to glucuronic acid in liver to form conjugated (direct) bilirubin
Direct bilirubin excreted to intestine
Total Bilirubin = conjugated + unconjugated
Pathology
Elevated In:
Hepatitis
Cirrhosis
Hemolytic Disorders (to include HDN*)
Enzyme Deficiencies
Hepatic Obstruction
*Hemolytic Disease of the Newborn
Hemoglobin Molecule
Heme converted to bilirubin
Red Blood Cell
Spleen
Unconjugated
Bilirubin
Bilirubin is conjugated to glucuronic acid
Liver
Conjugated or Direct
Bilirubin excreted into
Intestine for elimination
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6. BiliT Reaction Methodology
Methodology: Diazonium Salt/Diazonium ion w/blank
Equation:
Surfactants
Total Bilirubin + 2,4-Dichlorophenyl Azobilirubin
diazonium salt
Reactive Ingredients:
Surfactants 2,4-dichloroaniline
HCl HCl
Sodium Nitrite
Surfactant
Improved R1 surfactants result in decreased interference from hemolysis R1 R2
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7. BiliT Sample Handling Specimen Requirements: Serum Plasma
With or without gel barrier
Glass or plastic tubes
Venipuncture or capillary tube
Plasma
Lithium Heparin (with or without gel barrier)
Sodium Heparin
EDTA
Not Recommended: (potential for hemolysis)
Sodium Fluoride
Potassium Oxalate
Sample volume = 2.6 µL
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8. BiliT Sample Handling Specimens should be protected from bright light.
Bilirubin is photolabile.
Specimen storage:
Stored specimens must be inspected for particulates. If present, mix and centrifuge the specimen to remove particulates prior to testing.
Temperature
Maximum
Storage
20 to 25°C
2 to 8°C
-20°C
-80°C
1 day
7 days
6 months
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9. BiliT Reagent Handling
2 Reagent Kit Configurations
6L45-21
6L45-41
R1: 10 X 53 mL
R1: 8 X 93 mL
R2: 10 x 17 mL
R2: 8 x 28 mL
2750 tests/kit
3840 tests/kit
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10. BiliT Reagent Handling
Reagent is liquid, ready-to-use, two reagent kit.
Storage 2 to 8°C.
Total bilirubin reagent is light sensitive.
Unopened kit is stable until the expiration date when protected from light.
Store reagent cartridges in the reagent box when not on the System.
The reagents are susceptible to the formation of foam and bubbles.
Do not invert reagent cartridges prior to use.
Remove any air bubbles with a new applicator stick, or allow bubbles or foam to dissipate at storage temperature prior to use.
Reagent onboard stability - 21 days
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11. BiliT Calibration Calibration stability is 14 days.
Abbott Bilirubin Calibrator
Bilirubin Calibrator, E66-04, has not been revised or altered in relation to this reduction of sample volume.
Calibrator set name: Bil
REF
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C8000 PN v.1

12. BiliT Calibration Curve -AEROSET
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13. BiliT Calibration Curve – ARCHITECT c8000
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14. BiliT Reportable Range
Reportable Range: 0.1 to 25.0 mg/dL (Conv) to µmol/L (SI)
Limit of Quantitation: < 0.1 mg/dL (Conv) < 1.71 µmol/L (SI)
Limit of Detection: mg/dL (Conv) µmol/L (SI)
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15. BiliT Interference
The new BiliT assay is more resistant to hemolysis interference.
Acceptability is < 10% or mg/dL, whichever is greater.
Lower Decision Level
Interfering
Substance
Interferent
Concentration N
Target
(mg/dL)
Observed %
Recovery
Hemoglobin
1000 mg/dL
2000 mg/dL 7
1.1
0.9
86%
80%
Intralipid
1.0
1.2
1.4
115%
136%
Upper Decision Level
16.4
15.7
15.5
96%
95%
16.6
16.8
100%
101%
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16. BiliT Precision
CLSI protocol NCCLS EP5-A2 was used to evaluate precision:
Total CV < 5%
Precision Results
LEVEL N
Mean
mg/dL
Within Run
Between Run
Between Day
Total SD
%CV 1 80
0.75
0.01
1.2
0.00
0.4
1.5
2.0 2
4.11
0.02
0.5
0.03
0.8
0.05
1.1
0.06
1.4 3
5.86
1.0
0.07 4
15.70
0.3
0.08
0.12
0.16
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17. BiliT Method Comparison
Correlation per CLSI protocol NCCLS EP9-A2. The Total Bilirubin assay on an ARCHITECT cSystem was compared with the Roche Total Bilirubin assay (liquid 2,5-dichloro-phenyldiazonium tetrafluoroborate) on the Hitachi 917 Analyzer.
Adult
ARCHITECT vs. Hitachi
c8000 vs. c16000 N
138
Y-Intercept
0.10
-0.04
Correlation Coefficient
0.999
1.000
Slope
1.02
1.01
Range (mg/dL)
0.1 to 23.5
Neonatal N 54 53
Y-Intercept
0.32
0.02
Correlation Coefficient
0.997
1.000
Slope
0.99
1.00
Range (mg/dL)
1.0 to 19.1
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18. BiliT Assay Specific Information
Assay Method: Diazonium Salt
Reagent LN: 6L45-21, -41
Calibration:
Linear
Bilirubin Calibrator 1E66
Assay Stability:
Onboard stability: 21 days
Calibration stability: 14 days
Sample and Reagent Volumes:
Sample: L
R1 Reagent: L
R2 Reagent: L
Acceptable specimen:
Serum and plasma (heparin & EDTA), protect from bright light
Precision: Total CV< 5%
Reportable Range:
0.1 to 25.0 mg/dL (1.8 – mol/L)
LOD: mg/dL (0.86 mol/L)
LOQ: < 0.1 mg/dL (< 1.71 mol/L)
Reagent Considerations:
Light sensitive
Susceptible to formation of bubbles and foam
On AEROSET, BiliT must be line separated from:
6L35 MULTIGENT® Amikacin
6L31 MULTIGENT Quinidine
6E44 MULTIGENT Vancomycin
REF
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C8000 PN v.1

19. BiliT Troubleshooting Tips
Issue: QC has shifted with new cartridge of reagent, but the calibration interval has not expired.
Response: Confirm unused reagents are stored in the closed carton until ready for use. Within lot variability may be caused by exposure of the reagent cartridges to light, before they are placed on the system.
Issue: New reagent has foam and/or bubbles.
Response: If fresh reagent usually has foam or bubbles, confirm that customer is not inverting or shaking the reagent cartridge prior to use.
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20. BiliT Troubleshooting Tips
Issue: Specimen or QC material with a very low concentration does not achieve the 5% CV precision claim.
Response: As the concentration of specimens or QC material approaches the LoQ (limit of quantitation), by definition the %CV approaches 20%. The precision claim was developed around the bilirubin concentrations most often observed in routine testing. See the representative precision data for examples.
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21. BiliT Troubleshooting Tips
Issue: All (or the majority) of the samples on a run display results of < 0.1 mg/dL (1.71 mol/L) or an absorbance flag.
Response: This is probably due to foaming or bubbles. Inspect the reagent cartridge and remove any bubbles found per the instructions in the reagent package insert.
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22. Questions and Answers
Question 1: Is the intended use the same for the current Total Bilirubin G62 and this new product L45?
Answer 1: No, this new product L45 has claims for both adult and neonatal specimens.
Question 2: Is the product performance with neonatal specimens approved by my respective regulatory agency?
Answer 2: Yes. In the United States, the 510(k) submission for this product was cleared by the FDA with a neonatal claim. We have included the data to support the use of neonatal specimens with our international submissions.
REF
REF
REF
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23. Questions and Answers
Question 3: What makes this assay acceptable for use with neonatal samples?
Answer 3: Improved R1 surfactants result in decreased interference from hemolysis.
Question 4: Can’t all Total Bilirubin products with acceptable hemolysis interference be used on neonatal specimens – what makes the Abbott product different?
Answer 4: Many products do not specifically claim or disclaim the use of neonatal specimens. This product has been evaluated with neonatal specimens and the data to support the performance with these specimens was included in our regulatory submissions.
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24. Questions and Answers
Question 5: Do I need to take any special precautions to protect the reagent from light when the reagent cartridges are on the system?
Answer 5: No. Follow the instructions in your instrument manual for proper loading of reagent cartridges and use of the reagent supply center cover. Also, reagents are filled in brown wedges.
Question 6: Since the reagents are sensitive to light, can I store them in a lab refrigerator (2-8°C) with a glass door?
Answer 6: Yes, as long as you keep the reagent cartridges in the closed carton to prevent exposure to light.
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C8000 PN v.1

25. Questions and Answers
Question 7: Can I still use my reagents if they have changed color?
Answer 7: Yes, recalibrate and run controls.
Question 8: Can the reagent cartridge be placed on the system right after shipment arrival or if it is dropped?
Answer 8: The reagent is prone to formation of bubbles
and/or foam. Refer to the package insert for reagent handling and removal of bubbles. If a reagent cartridge is dropped you may wish to let it sit at 2-8°C to allow bubbles to dissipate and minimize volume depletion.
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26. Questions and Answers
Question 9: If the LOQ is < 1.71 mol/L (SI units), why is the low end of the reportable range set at 1.8 in the assay parameters?
Answer 9: The linear low value is the LOQ rounded up to the number of decimal places defined in the decimal places parameter field.
Question 10: I’m using the correct parameter file. Why isn’t the system recognizing the new Total Bilirubin reagent?
Answer 10: The default reagent names in the assay parameters are for L If you are using L45-41 change Reagent 1 name to BILIT61 and Reagent 2 name to BILIT52.
REF
REF
NOT FOR CUSTOMER DISTRIBUTION – INTERNAL USE ONLY For informational use only unless accompanied by the associated Lotus Notes form that contains electronic signatures.
C8000 PN v.1