INTRODUCTION

This publication covers the following topics: bilirubin metabolism, jaundice in the neonate, therapy, action limits for therapy, use of action limits, analytical requirements to the measurement of bilirubin, bilirubin measuring systems in general and a discussion.

BILIRUBIN METABOLISM

The major part (80 %) of bilirubin is produced by the degradation of the heme group of hemoglobin. The rest is derived from erythrocyte precursors or from the degradation of other heme-containing proteins. 

Hemoglobin is released from the erythrocytes and metabolized into bilirubin. Bilirubin is then transported to the liver by albumin. In the liver it is conjugated with glucoronic acid to form conjugated bilirubin which is then excreted via the bile ducts into the intestines and finally excreted with the feces. 

Small amounts are reabsorbed into the blood stream.

The following types of bilirubin are found in plasma:

• Unconjugated bilirubin – also called indirect bilirubin. It is poorly soluble in water and a high concentration of this is toxic due to its solubility in fatty tissue.
• Unconjugated bilirubin bound to albumin. It is water-soluble and non-toxic.
• Conjugated bilirubin – also called direct bilirubin. It is water-soluble and non-toxic.
• Delta bilirubin. It is covalently bound to plasma proteins, is water-soluble and non-toxic.

The ratio between conjugated and unconjugated bilirubin is usually constant and delta bilirubin is present in 0-2 %. It usually applies in neonates that total bilirubin = unconjugated bilirubin + conjugated bilirubin.

HYPERBILIRUBINEMIA IN THE NEONATE

60 % of all neonates develop jaundice during their first week of life due to factors such as:

• Natural rate of bilirubin production being greater per body mass compared with adults
• Shorter lifetime of the red blood cells
• Immature conjugating system in the liver
• Reduced bilirubin transport by albumin

Hyperbilirubinemia may in severe cases lead to kernicterus. Kernicterus is characterized by a deep yellow staining of various tissues in the brain, causing severe neural symptoms and abnormal developmental outcome. 

This should of course be avoided by all means by initiating correct therapy.

The introduction of phototherapy in the 1960s [1] virtually eliminated kernicterus for decades. 

Unfortunately it has reoccurred in many western countries in the last 5 years and is probably caused by a "why worry?” attitude amongst some neonatologists [2,3] in combination with an early discharge of mother and child after birth (see also Discussion).

THERAPY

In mild cases of jaundice, therapy may not be necessary. Since the early 1960s more severe jaundice has been treated with phototherapy, and in critical situations exchange transfusion is initiated.

The optimal phototherapy is obtained by exposing the neonate to blue light with a wavelength of 450 nm in a specially designed bed or blanket. 

When bilirubin absorbs the blue light, a photochemical reaction occurs which converts bilirubin to a water-soluble isomer which can then be excreted [4]. 

This type of therapy is relatively harmless. The major disadvantage is the disruption of the contact between the mother and the child and a mild dehydration.

Exchange transfusion is a very critical step to take and a decision of this has to be considered extremely carefully. 

The actual exchange transfusion is performed by taking out a small volume of the neonate’s blood in exchange of donor blood several times until the neonatal blood is close to being completely replaced. There is a relatively high risk of unwanted effects like fever, hemolysis, blood incompatibility, etc. 

Exchange transfusion is only performed in very critical situations where phototherapy has either been initiated too late or is insufficient.

ACTION LIMITS FOR THERAPY

The severity of hyperbilirubinemia is not only affected by the concentration of bilirubin, but also by other conditions such as

• Birth weight
• Gestational age
• Respiratory condition
• Acid-base status
• Immunization

However, decision on therapy is usually based on the measurement of total bilirubin together with a visual inspection of the color of the neonate.

Examples of action limits

Action limits are often established on an empirical basis. Below is an example of action limits based on age and another based on birth weight and condition.

From the American Academy of Pediatrics [5] in µmol/L (mg/dL) based on age:

TABLE I: units: µmol/L (mg/dL)

From the University Hospital in Copenhagen based on weight and condition:

TABLE II: 10 % of birth weight for bilirubin in µmol/L.

Use of bilirubin action limits

Due to the extensive use of action limits, accuracy and precision of the measurement of bilirubin are important.

The overestimation of bilirubin could potentially lead to unnecessary therapy. The side effects from phototherapy are not detrimental, but phototherapy may cause mild dehydration, rehospitalization if the mother and baby have been discharged and disruption of the contact between the mother and the baby.

In worst-case scenarios unnecessary exchange transfusion, which is a very aggressive treatment, is performed. As mentioned previously this entails the risk of fever, hemolysis, blood incompatibility, etc.

The underestimation of bilirubin could potentially lead to the withholding of necessary therapy and in a worst-case situation lead to kernicterus.

Both scenarios should by all means be avoided and one way to assure this is to have a reliable measurement of bilirubin so that it can justify the use of specific action limits.

ANALYTICAL REQUIREMENTS TO THE MEASUREMENT OF BILIRUBIN

The requirements to the measurement vary slightly depending on its use, e.g. whether it is for screening or for decisions on initiating, continuing or stopping therapy.

According to many clinicians a total error of ±10 % is acceptable with less strict requirements, when bilirubin is below 100 µmol/L (5.8 mg/dL).

Below are examples of analytical requirements for total bilirubin from the literature:

• Analytical goal as % CV derived from all known data on biological variation in healthy individual: 11.3 % [6].
• HCFA proficiency testing grading criteria for acceptable performance: Target value ±3.0 mg/L or 20 %, whichever is greater [7].
• Proposed quality specifications for total analytical imprecision as % CV: 11.3 %, and inaccuracy as % maximal allowable deviation: 9.8 %. These should be achieved in at least 95 % of experiments performed [8].
• Estimates of allowable imprecision from clinical and analytical outcome criteria with a decision level of 137 µmol/L (8 mg/dL):
  - Maximal allowable clinical imprecision: 11.2-15.7 %
  - Maximal allowable analytical imprecision: 9.7-13.0 % [9]
• Guidelines for providing quality STAT Laboratory Services Analytical Goals:
  Bilirubin at a level of 342 µmol/L (20 mg/dL): SD 9 µmol/L (0.5 mg/dL), max. bias: 17 µmol/L (1 mg/dL) [10].
• National Academy of Clinical Biochemistry, Standards of Laboratory Practice: Some clinicians feel that a total analytical error of ±10 % (approximately ±2 SD) is necessary for clinical needs [11].

Please note that in some of these references it is not stated to which age the requirement relates.

BILIRUBIN MEASURING SYSTEM IN GENERAL

Do the bilirubin measuring systems available fulfill the above listed criteria? The answer is unfortunately not always yes.

The different bilirubin measuring systems available on the market can be divided into:

• Transcutaneous devices
• Bilirubinometers
• Chemistry analyzers
• Whole-blood bilirubin analyzers

These may report a qualitative result, a semi-quantitative or a quantitative result, and this should of course be taken into consideration.

Bilirubin performance studies in the literature

It is well-known within the clinical chemistry community that large variations exist in the measurement of bilirubin from one system to another. Quote: "In 1960, 

Mather stated that the bilirubin determinations are perhaps the most notoriously unreliable of any in clinical chemistry. Twenty-two years later, Watkinson et al came to the same conclusion" [12] and this is also the conclusion of Newman et al [3] in 1992. 

A study conducted by Vreman et al [13] in 1995-96 reveals that only minor improvements have been achieved since 1960.

The study by Vreman et al was a very extensive study over eight months with 14 laboratories participating and representing three different measuring systems.

A lyophilized bovine bilirubin specimen of three different levels was measured. Table 3 shows mean lowest and highest results reported by the 14 clinical laboratories over eight months.

The within-laboratory variability across time, expressed as CV in % was:

• 1.3 % to 15.8 % for the 38 µmol/L (2.2 mg/dL) level standard
• 1.4 % to 15.4 % for the 69 µmol/L (9.9 mg/dL) level standard
• 2.0 % to 17.2 % for the 253 µmol/L (14.8 mg/dL) level standard

The authors conclude:

• The measured values remained fairly constant during the study at each laboratory.
• There was a very large variation from one analyzer to the other, even on the same type of analyzer.
• Because the within-laboratory variability is relatively low, the real problem exists between laboratories, even between those using the same brand of instrument, perhaps because of inconsistent calibrations locally.
• Matrix effect may explain some of the method-specific biases; however, on the same types of instrument this should be expected to be more or less constant. This suggests more or less serious calibration problems in the field.
• Until widely available standardization processes free of matrix effects are introduced, accurate and precise measurements in the clinical laboratories cannot be assured and this fact must be taken into consideration, especially when pediatricians are mandated to respond to proposed bilirubin action guidelines.
• The intralaboratory variability may have consequences for clinical practice on a case-by-case basis.

Other publications support this conclusion, e.g. Röhle et al [14]:
"Considering the special importance of bilirubin determination for neonatology, the state of analysis today is unsatisfactory. Substantial improvement could probably be brought about by means that are already available or could, in all probability, shortly become available."

Limitations of use and interference studies

The problems with poor performance of bilirubin measuring systems can probably to some degree be explained by either limitations of use or interfering substances.

Examples of limitations of use:
In their manual, a manufacturer of bilirubin measuring systems recommends that specimens from neonates less than 14 days old should not be analyzed for tBil because differences of ±10 % will be observed across the concentration range; however, a study by Langbaum does not support this conclusion [15].

Other limitations of use are, e.g. race, birth weight, etc. [16].

Examples of interference:
Various studies have been conducted on the interference from the presence of hemoglobin on the measurement of bilirubin on plasma. A major study was conducted by Grafmeyer et al [17] where 15 different analyzers measuring bilirubin were tested. Only three of these did not suffer from interference; the rest showed an interference in the range of –49 % to +73 % (–43 %, +55 %, –60 %, +47 %, +33 %, +36 %, etc). 

One type of analyzer showed interference from –43 % to +55 %. The hemoglobin concentration tested was 0.24 µmol/L (0.39 g/dL), which for a neonate corresponds to a hemolysis of 2 %. However, it should be noted that the range of bilirubin concentration tested was only 40-50 µmol/L.
 
Similar results are observed in a study conducted by Brady et al [18] and Scott et al [19].

DISCUSSION

It does not seem as if there will be any new therapy for neonatal jaundice in the near future. It is, however, clear that the current practice has to be reevaluated. There has been a reoccurrence of kernicterus in the last decade in the USA and other western countries [24]. 

In Denmark there have been six cases of diagnosed kernicterus from 1994-1998 after 20 years with none [25]. In the USA there have been 41 known cases of kernicterus from 1980-2000. 31 of these have been after 1990, representing a dramatic increase [26].

There are various reasons for this reoccurrence and some of the suggested actions to overcome this are:

• Changing the "why worry?" attitude that has been growing
• Introducing screening of all newborn
• Lowering the action limits for therapy
• Etc. [24,25]

With the information given above about the reliability of bilirubin measuring systems and the reoccurrence of kernicterus it could be worthwhile (re)evaluating whether the bilirubin measuring system in use at each institution provides the requested information and whether it supports the actual action limits used.

For healthy term newborns it may be sufficient with an indication of the actual bilirubin concentration, e.g. by a transcutaneous method, whereas preterm neonates, term neonates with respiratory problems, etc. require a more accurate information on the bilirubin concentration, as minor differences may be important. There is also some evidence that acid-base status [1,27,28] and hypoxia [29] affect the toxicity of bilirubin. 
  
It is not enough to strive for getting an accurate and precise bilirubin determination that can justify the use of specific action limits together with an evaluation of the general status. The systems chosen for bilirubin measurement may have a direct (negative) impact on the patient care. 

The sample volume is probably the most critical factor for bilirubin measuring systems. Besides that, the results from a bilirubin measurement should be back in due time to take appropriate action. 

There should be readily access to a measurement both from a time and from a logistical point of view. It should also be possible to measure bilirubin together with other parameters, e.g. the blood gases that can influence the decision on therapy [1,27,28].

CONCLUSION

Experience shows that Mather’s statement from 1960 about determinations perhaps being the most notoriously unreliable of any in clinical chemistry may to some degree still be the case.

With this in mind and along the fact about reemerging kernicterus it may be worthwhile that each institution carefully
(re)evaluate the performance of the actual method of measurement of bilirubin against current procedures, action limits, etc.  

When new methods are taken into use they should be evaluated carefully along with the clinical requirements, action limits, sample volume, limitation of use, turnaround time and other parameters that the result can be reported together with.