Direct Bilirubin 700nm

- - - - Ready-to-Use Room-Temperature-Stable Mono Reagent
      - Optimized for the Measurement of Conjugated Bilirubin
      - in the Presence of High Levels of Unconjugated Bilirubin
      - Minimal Chromatic Interference
      - Free from Indican Interference

Principles of the Reaction

The Synermed® Direct Bilirubin Reagent measures the absorbance of an oxidation product of bilirubin at 700 nm. The absorbance of the cholecyanin oxidation product is directly proportional to bilirubin concentration.

Conjugated bilirubin is oxidized in the presence of the electron transfer agent, KHC3, to a blue-green infrared light absorbing chromophore. Unconjugated bilirubin is not significantly detected due to the fact that it is not soluble under the conditions of the direct reaction mixture and remains bound to albumin.

KC3
conjugated bilirubin -> cholecyanin
(yellow) (blue-green)

Unlike traditional diazo methods, there is no chemical interference from hemoglobin in the reaction and hemoglobin has no absorbance at 700 nm. Lipemia has very little absorbance at the 700 nm wavelength used for the detection. Urinary indicans form no color in the reaction and do not interfere.

Limitations of the Procedure

The interference from hemoglobin was tested using the application to the IR200 and no interference was noted up to levels of 1000 mg/dL hemoglobin.
The interference from lipemia (tested as Intralipid) was determined using the application to the IR200 and no interference was noted up to levels of 1000 mg/dL lipemia.
The interference from a hemoglobin based blood substitute was tested and no significant interference was observed up to levels of 1000 mg/dL.
Urinary indican was tested at levels of 0.5 mM and 1.0 mM and no interference from indican was observed.

Special Performance Characteristics

This Synermed Direct Bilirubin methodology was correlated to a commercially available method. The calculated linear regression on 40 samples ranging from approximately 0 to 7.5 mg/dL with Synermed results on the Y-axis was: Y = 0.96X - 0.09 mg/dL with a correlation coefficient of 0.993.
The sensitivity of the procedure is such that an absorbance change of 0.001 will detect as little as 0.04 mg/dL when measured in a 1 cm light path.
The within run precision of the method as applied to the Synermed IR200 analyzer was determined by assaying samples of quality control materials at two levels of direct bilirubin concentration yielding the following results:

Mean SD CV

0.5 mg/dL 0.04 6.8%

5.9 mg/dL 0.06 1.0%
The run to run reproducibility of the method as applied to the Synermed IR200 analyzer was determined from the values obtained by 5 replicate analyses of quality control material assayed over a 20 batch period over 5 days. The following results were obtained:

Mean SD CV

0.5 mg/dL 0.03 5.2%

6.0 mg/dL 0.06 1.1%

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Cat. No.	Product Name	Packaging	Package Volume (mL)	Storage
IR750-X	Bilirubin, Direct 700 nm (Vet)	2x250 mL	500	0-4° C
IR750	Bilirubin, Direct	6x250 mL	3,000	0-4° C
IR750-911	Bilirubin, Direct	4x20 mL	80	0-4° C
IR750-WK	Bilirubin, Direct	3x55mL	165	0-4° C

Principles of the Reaction

Analytical Range

Limitations of the Procedure

Expected Values

Special Performance Characteristics

SDS

Product Packaging and Storage

Mean	SD	CV
0.5 mg/dL	0.04	6.8%
5.9 mg/dL	0.06	1.0%

Mean	SD	CV
0.5 mg/dL	0.03	5.2%
6.0 mg/dL	0.06	1.1%

Direct Bilirubin 700nm

Principles of the Reaction

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