Decreasing Matrix Effect On Diazo Reaction By Changing Sample Volume And Nitrite Concentration In To

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• Decreasing Matrix Effect On Diazo Reaction By
  Changing Sample Volume And Nitrite Concentration
  In Total And Direct Bilirubin Determinations

• Dr. Serkan Ahioglu
• Gata Haydarpasa Training Hospital Biochemistry
  Department ISTANBUL

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Bilirubin

• Orange-yellow bile pigment.
• Linear tetrapyrrolic structure
• Produced from protoporphyrine IX

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Bilirubin formation
Heme oxygenase
Biliverdin redüktase
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Bilirubin Fractionation by HPLC

• a unconjugated
• b mono-conj.
• g di-conj.
• d covalently bound to albumin

Ostrea EM, Ongtengco EA, Tolia VA, Apostol E. J
Pediatr Gastroenterol Nutr 1988 7 511-516.
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Methods Of Bilirubin Assays

• Direct Spectrophotometric Method
• Bilirubin concentration is directly
  determined by its absorbance at 454 nm HbO2
  interference is corrected by subtraction of
  absorbance at a second wavelength (540 nm)
• Enzymatic Method Bilirubin Oxidase
• Enzymatic oxidation of bilirubin to
  biliverdin and water reaction is monitored at
  405 to 460
• Reflectance Spectrophotometry
• Binding of bilirubin by a hydrophobic
  cationic polymer causes a shift in the spectrum
  of bilirubin the magnitude of the change
  as measured by reflectance photometry is related
  to bilirubin concentration
• High-performance Liquid Chromatography
• Methyl esters of conjugated and
  unconjugated bilirubin are detected at 430 nm
• Diazo Reaction

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• 1883 Diazo reaction first described by ERLICH.
• 1913 Bilirubin was first demonstrated to be
  present in normal serum by VAN DEN BERGH and
  Snapper.
• 1916 VAN DEN BERGH and Muller defined two forms
  of bilirubin The pigment that reacted in the
  absence of alcohol (accelerator) was termed
  direct. The pigment that required the presence
  of alcohol was termed the indirect bilirubin
  fraction.

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• Improved Method for Accurate Quantitationof Total
  and Conjugated Bilirubinin SerumJoel S. Novros,
  Thomas R. Koch, and Edward C. Knoblock. Clin.
  Chem. 25/11, 1891-1899(1979)

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• There is a lot of accelarators and modifications
  of diazo.
• Accelerator makes unconjuge bilirubin soluble and
  sets free from albumin bounds and breaks
  intramolekuler H bounds to make it polar.
• Methanol
• Cafein
• Urea
• Surface active agents
• Dimetil sulfoksit eg.
• So in Direct bilirubin measurements no
  accelarator used.

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• MalloyEvelyn
• METANOL is accelerator.
• is performed at pH 1.2 azobilirubin is measured
  at 560 nm
• susceptible to significant hemoglobin
  interference
• JendrassikGrof
• sodium benzoatecaffeine is accelerator.
• performed near neutral pH,
• but absorbance is measured at alkaline pH
  (approximately ph13) at 600 nm.
• Most commonly used method.
• Walters-Gerarde
• Dimethyl sulfoxide is accelerator.
• Interferences with Hb up to 500 mg/dl and
  trigliserides 2000mg/dl are negligible.

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• Reagent Total Bilirubin A
• Sulfanilic acid
  29 mmol/L
• HCl
  145 mmol/L
• Dimethyl sulfoxide
  6,36mol/L
• Reagent Direct Bilirubin A
• Sulfanilic acid
  29 mmol/L
• HCl
  145 mmol/L
• Reagent Total/Direct Bilirubin B
• NaNO3
  5,7mmol/L

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• We measured total and direct bilirubin levels in
  control materials (n120) and in patients with
  Gilberts disease (n37) and hyperbilirubinemia in
  the newborn (n23) by changing final nitrite
  concentrations between 0,01-1,5 mmol /L in the
  reaction mixture.
• The optimal nitrite concentration in the final
  reaction mixture was 0,1 mmol/L for direct and
  0,21 mmol/L for total bilirubin measurements.
• This concentrations were lower than the values
  (0,15 mmol/L and 1,6 mmol/L respectively)
  previously reported.

• Inaccurate Values for Direct Bilirubin with
  Some Commonly Used Direct Bilirubin Procedures
  Kwok- Mlng ChanMitchell G. Scott,2 Tal-Wing
  Wu,3Ray E. douse,4 David R. CaMn,5 John Koenig,5
  Don A.Uchtl,5 and Jack H. Ladenson CLIN. CHEM.
  31/9, 1560-1563 (1985)

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• To eliminate matrix effects on the bilirubin
  determinations, we added different detergents
  (triton X-100, Brij 35 and tween 20 ) into the
  total and direct bilirubin reagents.
• All detergent types decreased the reaction rate
  and prevented reaction to reach endpoint.
• So we did not use any detergent.

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• Normally, billirubin determinations need high
  sample/reagent ratio and this condition makes the
  effect of sample matrix higher.
• Thus we made numerous tries to find the
  sample/reagent ratios for total and direct
  bilirubin measurements.
• We found that the ratios of 4/260 for total and
  10/320 for direct bilirubin measurements, made
  the matrix effect minimum.

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• We adapted total and direct bilirubin
  determinations to
• Beckman Coulter LX-20 automated analyser,
• Beckman Coulter synchron CX-5 analyser,
• Olympus AU800 analyser,
• Abott Architect c8000 auto analyser.
• Total and direct bilirubin measurements were well
  correlated between all analysers by using
  different commercial controls.
• Beckman Coulter Decision Multilevels,
• Biorad controls,
• Roche controls,
• Olympus control
• Patient Samples
• The mean CV values were between 3.4 and 4.8
  for total and direct bilirubin measurements
  respectively

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• In conclusion, we have improved total and
  direct bilirubin measurement methods, which could
  not effected by sample matrix by changing
  nitrite concentrations and lowering sample
  volumes.
• THANKS