I'm working on a spectrophotometric clot formation and lysis assay and need to assess interference from bilirubin. I can't find a commercial high bilirubin control that will produce significant icterus (say bilirubin of at least 5mg/dl and preferably higher) when diluted 1 in 10 with normal plasma, and I don't have any patient samples in this range that aren't also hemolyzed. I've tried dissolving various weights of bilirubin in water with a drop of 1M NaOH and in DMSO, but it won't stay in solution. I think alcohol dilutions will just precipitate the plasma proteins.
Filtering this stock solution with a 0.2uM syringe filter does not remove the granular material. If I leave it settle and just draw off the upper relatively clear layer, not only do I not know the end bilirubin concentration, but the clot will form but not lyse which I think is due to the alkaline pH or DMSO rather than the bilirubin.
If anyone has any advice on how to dissolve bilirubin without inhibiting coagulation I'd be really grateful!
Have you looked for bilirubin calibrators or calibration verification materials that could be in this range? I'd look for these first.
This is not an ucommon problem in the lab.
I worked in the Clinical Lab at Mayo Clinic for 10 years. I recall them having a specific method to address this. I suggest you call Mayo Med Labs in Rochester. They have a Clinical Pathology resident on the "hot seat" 24/7, and this is the type of questions they are there to answer of if they don't know the answer they will look it up and get back to you the same day.
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Thank you all for your helpful suggestions. After playing around with a few different solvents and pHs, I gave up and doubled the strength of my calcium buffer so I could add larger volumes of a commercial calibration solution and thus achieve desired concentrations without excessive dilution of plasma. I will contact the Mayo and post their answer if they have a more elegant solution!
If you want to test only for the optical disturbances caused by bilirubin you can used potassium ferrocyanide instead. It has a similar absorption spectrum (max at 420nm) and a good solubility in water. It is a recommended test substance for the optics of the BCS coagulation analyzers (Siemens Healthcare).
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