Hello I hope that you guys are there are enjoying your SIP at ur individual places. I’m in the clinical biochemistry department for my SIP. And I am going to talk about bilirubin measurment for neonates. In my lab we use a bilirubinometer that is a direct spectrophotometer that measures total bilirubin in neonates( babies ) by using their serum samples. These samples are usually collected in capillary tubes as neonates do not have much blood to be drawn out for analysis.
Mechanics of the bilirubinometer
1. Light passes through the cuvette and is split by a dichroic mirror and directed to 460 and 550 photo detector
2. Output of the photometer is calculated by control electronics to measure total bilirubin
3. bilirubin and oxyhaemoglobin’s peak absorbance is 460nm. It is necessary to measure oxyhaemoglobin at 550nm The difference in reading at 460 and 550nm is the total bilirubin value.
4. usually haemolyzed samples do not affect the result as the bilirubinometer in my lab substracts the oxyhaemoglobin value from the total value that is collected by the photometer .
5. this bilirubinometer only measures neonate sample as interfering substances become more prevalent and will affect results for adults
Interfering substances
Pigments that are absorbed at 460nm will affect the reading of the sample in the bilirubinometer. Some examples are lipochromes and carotenoids. Neonatal samples that are collected usually lack such interfering substances to affect the reading
Certain drugs also interfer with readings, like for example sulfonamides, phenols and amines.
Intravenous fat emulsions and lipaemic samples are also know to affect the reading. Such an example is Lyposin that has affected direct spectrophotometric measurements
Daily Controls
2 assayed galss cuvette is provided by the manufacturer, it is usually done between runs, at the start of each day.
EQC
my lab has taken part in certain EQC programmes for bilirubinometer for neonates. Some examples are CAP, neonatal bilirubin assay programme
Materials
Sample
test tube
special pipette
centrifuge machine
cuvette
bilirubinometer
Methods
1.Place capillary tube sample into test tube affixed with plastic stopper to prevent blood from leaking out during centrifuging. This will cause of loss of serum which is precious as neonates are involved
2.place in centrifuge and counter balance
3.spin for 5 mins at 3500 rpm
4.using a capillary tube pipette, fill cuvette with serum. The cuvette volume is 20 uL but there’s no need to measure the sample as the exact volume is determined by the cuvette. Cuvette needs to filled full.
5.the outside surface of the cuvette must be free of serum, dust and smudges as it will affect the machine’s capability of interpreting the result
6.bubbles will affect the reading, thus the rectangular area of the cuvette must be cleared of bubbles. Tap lighting on cuvette to remove bubbles.
7.place cuvette in bilirubinometer and press start button.
Yuxuan
picture reference: www.reichertai.com
Mechanics of the bilirubinometer
1. Light passes through the cuvette and is split by a dichroic mirror and directed to 460 and 550 photo detector
2. Output of the photometer is calculated by control electronics to measure total bilirubin
3. bilirubin and oxyhaemoglobin’s peak absorbance is 460nm. It is necessary to measure oxyhaemoglobin at 550nm The difference in reading at 460 and 550nm is the total bilirubin value.
4. usually haemolyzed samples do not affect the result as the bilirubinometer in my lab substracts the oxyhaemoglobin value from the total value that is collected by the photometer .
5. this bilirubinometer only measures neonate sample as interfering substances become more prevalent and will affect results for adults
Interfering substances
Pigments that are absorbed at 460nm will affect the reading of the sample in the bilirubinometer. Some examples are lipochromes and carotenoids. Neonatal samples that are collected usually lack such interfering substances to affect the reading
Certain drugs also interfer with readings, like for example sulfonamides, phenols and amines.
Intravenous fat emulsions and lipaemic samples are also know to affect the reading. Such an example is Lyposin that has affected direct spectrophotometric measurements
Daily Controls
2 assayed galss cuvette is provided by the manufacturer, it is usually done between runs, at the start of each day.
EQC
my lab has taken part in certain EQC programmes for bilirubinometer for neonates. Some examples are CAP, neonatal bilirubin assay programme
Materials
Sample
test tube
special pipette
centrifuge machine
cuvette
bilirubinometer
Methods
1.Place capillary tube sample into test tube affixed with plastic stopper to prevent blood from leaking out during centrifuging. This will cause of loss of serum which is precious as neonates are involved
2.place in centrifuge and counter balance
3.spin for 5 mins at 3500 rpm
4.using a capillary tube pipette, fill cuvette with serum. The cuvette volume is 20 uL but there’s no need to measure the sample as the exact volume is determined by the cuvette. Cuvette needs to filled full.
5.the outside surface of the cuvette must be free of serum, dust and smudges as it will affect the machine’s capability of interpreting the result
6.bubbles will affect the reading, thus the rectangular area of the cuvette must be cleared of bubbles. Tap lighting on cuvette to remove bubbles.
7.place cuvette in bilirubinometer and press start button.
Yuxuan
picture reference: www.reichertai.com
10 comments:
Hi Yuxuan
May I know which part of the fetus is blood taken from, and why does neonatal samples ususally lack interfering substances to affect the reading?
Kudos
Johan
it's usually taken from the feet and its when the baby is already born, so its not fetus. and neonatal samples lack interfering substances because most of these substances are either not yet produced in the babies or the natural body lack them, like certain drugs.
Yu Xuan
i was just wondering, your QC materials are just the cuvettes or are they filled with some fluid you need to reconstitute 1st?
-cornelyus
hey yuxuan!
why do we measure for baby total bilirubin? is it same as adult to detect hepatic problems?
And is there any range for the values of neonate total bilirubin? what happen if it is not normal.. like do you repeat the test or do another confirmation?
thanks!
cheers,
huimin
tg01 =)
Yo YuXuan!
Some queries to ask:
1)What is a dichroic mirror? Why is dichroic mirror preferred over monochroic mirror?
2)How can Lyposin affect direct spectrophotometric measurements?
3)What temperature do you spin the neonatal blood at and why at that temperature?
4)Why neonatal samples are lacked of lipochromes and carotenoids and how can they affect the readings?
Hmmm, there is a typo error in your sentence. "Tap lighting on cuvette to remove bubbles".
Hope you can clarify my doubts...
Thankz alot! See you tomorrow!
Han Yang
TG01
Hey Yuxuan (:
Why does the absorbance reading range is different for bilirubin and oxyhaemoglobin?
As you said that bilirubin have to be measured at 460nm and oxyhaemoglobin at 550nm.
Another thing is, I have encountered problems when I am doing this test. My lab's method is to break the capillary tube in half near the point of separation of serum & red cells. And fill up the cuvette. But sometimes a bit of blood leaks into it, any idea will it cause any variation?
Yvonne Teo
hey yuxuan,
may i know what are some of the troubleshooting methods you carry out if the substances interefere with the readings and what are some of the precautions you have to take to minimise such incidents from happening?
thanks
Liyanah Zaffre
0607718D
TG02
yoz...
what is the reference range for the results?how do you deal with samples that has exceeded the reference range?
Ivan here.
Hi Yuxuan,
Don't mind can you rephrase or explain this sentence
"3. bilirubin and oxyhaemoglobin’s peak absorbance is 460nm. It is necessary to measure oxyhaemoglobin at 550nm The difference in reading at 460 and 550nm is the total bilirubin value."
Thanks
Jean Leong
TG02
To corn: the machine itself is calibrated. there's only the control to do every day.
yuxuan
to hui min: measuring bilirubin is to detect NNJ. neo natal jaundice. and if its usually above 200 on the meter we repeat it on the other machine. all values must be reported to the ward
to yvonne and jean: both haemoglobin and bilirubin can be measured together to give false positive results. thus you need to measure bilirubin n Hb at a level that bilirubin cant be measured for subtracting. and my lab uses a special pipette so we do not need to break the tube.
to Ivan: there is no reference range. all values must be reported
to Liyanah: most of the time babies do not have interfering substances unlike adult. that's why we use a different machine. till now i've not experienced a baby with interfering substances of great amount
to han yang: the temperature used to spin is room temp. but light must be avoided as bilirubin will break down under light. i'll get back to you on the other questions as i have changed labs. oh and babies do not have such intefering substances, as you only acquire them from eating certain substances, which baby have limited choice of food at the time.
Yuxuan
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