Using an automated chemistry analyzer, a blood sample yielded the following result:
- Total bilirubin 2.0 mg/dL
- Conjugated bilirubin 2.2 mg/dL
- Unconjugated bilirubin -0.2 mg/dL
How could this happen?
Some analyzers measure bilirubin using the Jendrassik-Grof method. In this method, sample is divided into two portions. The first portion is measured for conjugated bilirubin, while the second one is measured for total bilirubin. Both measurements use diazotized sulfanilic acid that will react with bilirubin, producing red azobilirubin of which intensity is proportional to the level of bilirubin. The difference between both measurements is that the one for total bilirubin uses additional compound containing caffeine.
In the first portion, diazotized sulfanilic acid reacts with conjugated bilirubin much faster than unconjugated bilirubin. In a set amount of time (e.g. 10 minutes), it is expected that all conjugated bilirubin will have reacted while the unconjugated one will not. Hence, all red azobilirubin will have come from conjugated bilirubin alone.
In the second portion, additional caffeine acts as an accelerator, enabling diazotized sulfanilic acid to react with unconjugated bilirubin, producing red azobilirubin in the same time window (10 minutes). As a result, the red azobilirubin produced will have come from conjugated and unconjugated bilirubin.
The level of unconjugated bilirubin is then calculated by substracting the level of total bilirubin with conjugated bilirubin.
Had the test mentioned in the case been done manually, we can assume that the peculiar result was caused by uneven portions, uneven test time, uneven exposure between the two portions (bilirubin is quickly broken down in the presence of light), or not enough caffeine. But, the test was done in an automated fashion, eliminating the problems caused by manual testing. So, as of right now, the question remains unanswered.
What can be done?
Since I don’t know how such result could happen, in case of encountering similar result, I can only suggest repeating the test.
UPDATE
It seems that for the analyzer that my laboratory uses (Cobas c510), this is caused by the close values of total and conjugated bilirubin (in this case, the difference is only 0.2 mg/dL). I've noticed this after seeing that every time the phenomenon happens, the difference is never large.
The explanation is due to the close values and coefficient of variation (CV), the total bilirubin value can sometimes hit a lower number than the conjugated bilirubin, resulting in a negative unconjugated bilirubin.
For a solution, an analyst in my laboratory once suggested to switch the values. It seemed absurd and very wrong back then. But now, I think it's plausible considering solely that both values are supposed to be close and within the range of each one's CV anyway.
- Total bilirubin 2.0 mg/dL
- Conjugated bilirubin 2.2 mg/dL
- Unconjugated bilirubin -0.2 mg/dL
How could this happen?
Some analyzers measure bilirubin using the Jendrassik-Grof method. In this method, sample is divided into two portions. The first portion is measured for conjugated bilirubin, while the second one is measured for total bilirubin. Both measurements use diazotized sulfanilic acid that will react with bilirubin, producing red azobilirubin of which intensity is proportional to the level of bilirubin. The difference between both measurements is that the one for total bilirubin uses additional compound containing caffeine.
In the first portion, diazotized sulfanilic acid reacts with conjugated bilirubin much faster than unconjugated bilirubin. In a set amount of time (e.g. 10 minutes), it is expected that all conjugated bilirubin will have reacted while the unconjugated one will not. Hence, all red azobilirubin will have come from conjugated bilirubin alone.
In the second portion, additional caffeine acts as an accelerator, enabling diazotized sulfanilic acid to react with unconjugated bilirubin, producing red azobilirubin in the same time window (10 minutes). As a result, the red azobilirubin produced will have come from conjugated and unconjugated bilirubin.
The level of unconjugated bilirubin is then calculated by substracting the level of total bilirubin with conjugated bilirubin.
Had the test mentioned in the case been done manually, we can assume that the peculiar result was caused by uneven portions, uneven test time, uneven exposure between the two portions (bilirubin is quickly broken down in the presence of light), or not enough caffeine. But, the test was done in an automated fashion, eliminating the problems caused by manual testing. So, as of right now, the question remains unanswered.
What can be done?
Since I don’t know how such result could happen, in case of encountering similar result, I can only suggest repeating the test.
UPDATE
It seems that for the analyzer that my laboratory uses (Cobas c510), this is caused by the close values of total and conjugated bilirubin (in this case, the difference is only 0.2 mg/dL). I've noticed this after seeing that every time the phenomenon happens, the difference is never large.
The explanation is due to the close values and coefficient of variation (CV), the total bilirubin value can sometimes hit a lower number than the conjugated bilirubin, resulting in a negative unconjugated bilirubin.
For a solution, an analyst in my laboratory once suggested to switch the values. It seemed absurd and very wrong back then. But now, I think it's plausible considering solely that both values are supposed to be close and within the range of each one's CV anyway.
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