Critically evaluate the methods in general use for the ...



Critically evaluate the methods in general use for the measurement of bilirubin and its fractions in serum or plasma. (March 2003, paper 1, question 1a)

Examiners comments:

There were some good answers to this question, although some lost marks giving the full clinical biochemistry of Bilirubin, which was not asked for.

Plan:

- Bilirubin & its fractions

- Methods used in the lab

▪ Diazo- TBil and DBil

• Long term stability

• Acceptable transferability

• No universal calibrant

• Hb interference- addition of ascorbate reduces

• Turbidity- caffeine or surfactants, or bichromatic blanking

▪ Spectrophotometric- dry slide

• (-bilirubin not measured, but can be calculated

• Interference with Hb, carotenoids and turbidity

▪ Oxidase

• Good agreement with Jendrassik & Grof

• All 4 fractions measured in TBil

• Protein binding reduces the reaction rate- detergent releases protein bound Bilirubin

• Hb interference reduced by EDTA

• DBil- depends on pH used- pH 10 = 5% unconjugated is measured as conjugated. pH 4ish = conjugated & (-bilirubin

- PoC methods

▪ Spectrophotometric- bilirubinometer

• Different Bilirubin species have different absorbance spectra & interfering compounds have range of overlapping absorbance- wavelengths used are a compromise

▪ Spectrophotometric- transcutaneous bilirubinometers

• Bilirubin cannot be accurately predicted

• Cannot be used in patients undergoing phototherapy

• Non-invasive, instant

• Removes the visual assessment of jaundice

• Portable

• No IQC or EQA

▪ Spectrophotometric- BGA

Bilirubin is the orange-yellow pigment derived from senescent red blood cells. It is metabolised mainly in the liver and excreted in bile and urine.

4 bilirubin fractions have been identified

1. free bilirubin ((-bilirubin)

2. protein bound bilirubin ((-bilirubin)

3. mono-conjugated bilirubin ((-bilirubin)

4. di-conjugated bilirubin ((-bilirubin)

Samples taken for measurement of bilirubin should be protected from light and also kept at a low temperature because bilirubin is photoliable when exposed to white or UV light, and the stability of bilirubin is also temperature dependent.

The most widely used methods to measure bilirubin as demonstrated by participants of UK NEQAS bilirubin scheme are

– Diazo dye binding (~60%)

– Spectrophotometry (~10% dry slide)

– Oxidation by bilirubin oxidase or potassium ferricyanide (~26%)

Diazo-dye assays

Total bilirubin

In this reaction diazotised sulphanic acid reacts with bilirubin to produce 2 azodipyrroles which are reddish/purple at neutral pH.

Bilirubin + diazonium azobilirubin

ion

At acid pH, the reaction with conjugated bilirubin is very rapid and complete within 2 minutes but (-bilirubin reacts more slowly. The reaction with unconjugated pH is very pH dependent and the rate falls off rapidly in acidic conditions. To reduce this pH dependence, a number of compounds have been used to increase the rate of diazotisation (methanol, caffeine, sodium benzoate). The method described by Jendrassik & Grof uses caffeine and sodium benzoate and detects unconjugated and conjugated bilirubin.

Most methods used in the clinical chemistry lab are based on that of Jendrassik & Grof.

The method used in my lab, is the Roche total bilirubin liquid stable assay. This uses a method uses a method developed by Wahefeld, in which a surfactant is used to accelerate the reaction.

The accelerants used also remove the unconjugated protein bound bilirubin from its association sites on albumin.

The diazo methods generally use a dichlorophenyl diazonium substrate. These have relatively long term stability, are available in ready to use solutions and can be kept on board automated analysers in sufficient volumes for days to weeks.

The diazo methods have acceptable transferability between labs and produce reliable and reproducible results, quickly. These assays are simpler to standardise but there is no universal calibrant is available at this time.

A major problem is haemoglobin interference due to both direct spectral interference and also oxyhaemoglobin mediated breakdown of bilirubin both during and after diazotisation. The interference by haemoglobin is sensitive to variation in reagents, dilutions, reaction times, bilirubin concentration, the wavelengths used, pH and diazonium salt used.

The addition of ascorbic acid is often included in diazo assays. Ascorbate reduces the instability of the azobilirubin in the presence of haemoglobin.

Ascorbate also destroys the diazo reagent and stops the coupling reaction. It is therefore added to the reaction mix after the reaction is complete.

The interference by turbidity is decreased through the use of caffeine or surfactants that have clearing properties. The use of biochromatic blanking also reduces the effect further and also that if interference from other serum constituents.

Conjugated

Conjugated bilirubin is the mono- and di-glucuronides plus any other minor conjugates. It is the same assay as for total bilirubin except that the compound that accelerates the reaction and also solubilised the unconjugated bilirubin from protein is not included in the reaction mix.

Spectrophotometry

Total and conjugated bilirubin

Caffeine with a surfactant is used in the spreading layer of a dry-film spectrophotometric method for bilirubin. The bilirubin in the sample is exposed to a binding agent at pH 8.0 and the light reflected from the slide is measured at 2 wavelengths to allow for the 2 different maxima of the unconjugated (420-425nm) and conjugated (460-465nm) species. This method uses unconjugated and conjugated bilirubin and therefore total bilirubin, but not (-bilirubin.

The (-bilirubin can be calculated by subtracting unconjugated and conjugated bilirubin from the total bilirubin.

Interference in these methods arise from any other serum components which absorb light at the chosen wavelengths. In practice, problems arise with regard haemoglobin, carotenoids and turbidity. For most methods the wavelengths used are chosen to minimise haemoglobin interference.

Enzymatic

Total

Based on the oxidation of bilirubin with bilirubin oxidase to biliverdin with molecular oxygen. Near pH 8 and in the presence of sodium cholate and SDS, all 4 fractions are oxidised to biliverdin which is further oxidised to purple and finally colourless products. The decrease in absorbance at 425nm or 460nm is proportional to the total bilirubin concentration. This method has good agreement with the Jendrassik and Grof methods.

Protein binding reduces the reaction rate. Addition of an anionic detergent releases protein bound unconjugated bilirubin. Inclusion of EDTA reduces any effect of haemoglobin.

Conjugated bilirubin

Conjugated bilirubin is measured at a pH 3.7 to 4.5

At this pH, the enzyme oxidises bilirubin conjugates and (-bilirubin, but not unconjugated bilirubin (protein bound).

At pH 10 the enzyme only oxidises conjugated bilirubin and not (-bilirubin. However, 5% of unconjugated bilirubin is measured as a conjugated with this method.

Point of Care Methods

Spectrophotometric- Bilirubinometers

These are still used on some neonatal wards although the availability of bilirubin on blood gas analysers is reducing their use.

In most methods, 2 absorbances are measured. 1 near the bilirubin peak- when bilirubin is present, the absorbance increases at 454nm and is proportional to the bilirubin concentration. The second absorbance is made at a higher wavelength as a blank against a number of possible interfering compounds- subtraction of absorbance at 540nm corrects for oxyhaemoglobin.

Since different bilirubin species have different absorbance spectra and the interfering compounds have a range of overlapping absorbance, both peak and blank wavelength are compromises, which have generally been arrived at empirically.

To improve on the diversity found with these methods, the use of a diluent containing caffeine- which also improves any interference from haemoglobin).

However, this limits their suitability for use on neonatal wards.

Blood gas bilirubin

Capillary bilirubin is measured using spectrophotometry with multiple wavelength readings and algorithms to correct for haemoglobin, foetal haemoglobin and lipaemia.

Transcutaneous measurement of bilirubin

Non-invasive approach. Reflectance densitometry which uses sophisticated wavelength filtering and algorithms to correct for skin colour and convert the raw absorbance readings to (mol/L for bilirubin.

However, the bilirubin levels cannot be accurately predicted using these machines- wide CVs found in comparison with lab bilirubin and may underestimate bilirubin at concentrations above 170(mol/L. The variability in readings may be due to the site of sampling, skin colour, degree of prematurity and rate of change in plasma bilirubin.

This method cannot be used to monitor infants undergoing phototherapy.

It is however, it is non-invasive and provides instantaneous results. It also removes the visual assessment of jaundice and therefore may be useful in screening newborn infants before discharge. It also reduces the number of heel sticks performed- good for child and the family and saves money. It is portable and may be used by community nurses.

There is no IQC other than electrical checks and no EQA schemes available.

Other methods

Rarely used in routinely such as HPLC or mass spectrometry.

HPLC can detect and separate the bilirubin photoisomers produced by phototherapy. Clinically, offers little if any aid to the physician in the differential diagnosis of jaundice. Furthermore, HPLC is not a reference method because its precision and accuracy is currently inadequate.

Urine bilirubin

Only conjugated bilirubin us excreted in the urine, therefore, its measurement is an indirect test for the increased concentration of conjugated bilirubin in serum. The commonest method is the use of a dipstick impregnated with diazo reagent. It turns pink to red/violet colour in the presence of bilirubin. Its intensity is proportional to the bilirubin concentration.

Simple, quick and inexpensive

PoCT

Specific and has a low incidence if false positives

Subject to interference from certain medications

Strip must not be in the urine for greater than 1 second.

References

Ann Clin Biochem 1991; 28: 119-130

Ann Clin Biochem 2008; 45: 452-462

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Conjugated fraction

Considered together as the un-conjugated fraction

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acid

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