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Gladys Chia
Chew Yu Mei
Yvonne Teo
Cornelyus Ng
Lau Leslie
12 September 2008
10:28:00 PM
This week, I am going to share with you guys about one of the online analyzer that I have in my lab, the DxI. The full name of this analyzer is Beckman Coulter Unicel DxI 800 Access Immunoassay System. The tests that are conducted on DxI in my lab are Ferritin, Folate, Vitamin B12, Cortisol, Testosterone, DHEA-S, Intact PTH, Insulin, Ostase, Free T3, Total T3, Free T4, Total T4 and TSH.
As what I have mention, DxI is an online analyzer, which means it’s connected to the LAS, therefore there are limited things to do (not much hands on) because most tests will be run automatically. What I did in this section is the daily maintenance like backing up the data, running special clean solution, toping up of reagents, wash buffer, reaction vessel, substrate, running of calibration and controls. However, there are 2 “special” tests that require pre-treatment before loading them onto the analyzer. They are red blood cell folate and urine free Cortisol. But, I am going to only describe how urine free Cortisol is done.
Urine Free Cortisol
Cortisol is the major glucocortisol produced and secreted by the adrenal cortex. It affects the metabolism of the protein, fat and carbohydrates, the maintenance of muscle and myocardical integrity and the suppression of inflammatory and allergic activities.
Cortisol bound to protein is protected from the metabolism by the liver. Unbound or free Cortisol in the serum is metabolized by the liver resulting in a wide variety of forms or metabolites. Many of these metabolites (conjugated, glycuronide and sulfate forms) are water-soluble and rapidly voided in the urine. A small amount (<100ug/24h) of Cortisol and other extractable metabolites are also excreted in the urine. Urine Cortisol can be measured by performing an extraction step which removes some of the water-soluble metabolites prior to analysis or by analyzing urine directly. Immunoassays measure urine Cortisol as well as some immunoactive metabolites, therefore, representative expected value should be established for each immunoassay method. The measurement of urine Cortisol reflects the amount of unbound or free serum Cortisol and aids in the diagnosis of adrenal hyperactivity. An elevated level of urine Cortisol is considered diagnostic for Cushing’s syndrome (adrenal Cortisol hyperfuction)
As urine free Cortisol (UFC) is as often ordered by the doctors, my lab only runs it twice per week. We’ll do the treatment of the samples on every Monday and Thursday and the samples will be run on every Tuesday and Friday. The average amount of sample accumulated is around 5 specimens. The controls (which require per-treatment) are only run on days when UFC is run.
Urine Cortisol Treatment Procedure
1. Thaw 2 sets of control (level 1 & 2) from the freezer.
2. Label 2 glass tubes for each test urine and label 1 glass tube for each control.
3. Centrifuge all aliquots of 24 hour urine at 3500rpm for 5 minutes.
4. Pipette 500ul of the clear supernatant from the test urine into the first glass tube.
5. Add 500ul of ethyl acetate into each test urine/control.
6. Vortex vigorously for 60 seconds. ‘
7. Centrifuge at 2500rpm for 5mins.
8. Pipette 200ul from the ethyl acetate layer (top) into the second glass tube.
9. Leave tubes to dry overnight in the fume cupboard.
Urine Free Cortisol Run Procedures
1. Check that ALL glass tubes are dry.
2. Pipette 200ul of SO calibrator into one set of the controls. Vortex well.
3. Analyse Cortisol on the DxI. (Ensure that control values are within acceptable limits.
4. Pipette 200ul of SO calibrator into each test urine. Vortex well.
5. Pour this mixture into cups & run them on the DxI requesting for Cortisol.
Note:
- While handling ethyl acetate, always make sure it’s in the fume hood because is has an unbearable smell.
- Glass tubes must be used for this test because the reaction will blur the surface of plastic tube, therefore not allowing us to have a clear visualization of the two layer.
- If the top layer of ethyl acetate is not properly pipetted out (mixing with the bottom layer), the overnight specimen will not dry up, leading to a unsuccessful treatment and therefore we have to re-do the whole thing.
http://www.beckmancoulter.com/products/instrument/immunoassay/UniCel_DxI_800.asp
UniCel® DxI 800 Access® Immunoassay System
Hope you guys understands what i have mentioned.
Thanks!
Chew Yu Mei
TG01
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