A sample blank can correct for potential error from existing color or turbidity in the sample before reagents are added.

when Unconjugated (“indirect”) bilirubin reach toxic levels (18-20 mg/dL) it can transfer through Blood Brain Barrier

There are three types of bilirubin: total, direct (conjugated),and indirect (unconjugated).
Total bilirubin is composed of the direct bilirubin plus
the indirect bilirubin. The total bilirubin level increases with any type of jaundice.
direct or conjugated, bilirubin is excreted by the gastrointestinal tract, with only minimal amounts entering the bloodstream. It was originally named direct” bilirubin because this water-soluble type of bilirubin reacts directly with the reagents added to the blood sample(old detection method). Its level rises in the blood when obstructive jaundice (as from gallstones) or hepatic jaundice occurs, because the bilirubin is unable to reach the intestines for excretion and instead, enter the bloodstream for excretion by the kidneys.

Indirect bilirubin, also known as free or unconjugated bilirubin, is normally found in the bloodstream. Its name comes from the fact that this non-water-soluble bilirubin does not directly react with reagents added to a blood sample. Alcohol must be added for the reaction to occur(old detection method).
Indirect bilirubin rises in cases of hemolytic jaundice, in which the breakdown of hemoglobin results in a higher than normal level of indirect bilirubin being present in the bloodstream. This is the type
of bilirubin elevated in cases of hepatocellular dysfunction, such as hepatitis.

In the van den Bergh reaction, hemolysis in serum samples results in decreased azobilirubin color development and hence in measured bilirubin levels which are falsely low. The reduction in measured bilirubin content of serum samples ranged from 5 to 15 per cent at discernable levels of hemolysis depending on the volume of serum used in the reaction. No adequate explanation for the effects of hemolysis on the van den Bergh reaction could be found

Also known as Ionized calcium.It is the metabolically active form of calcium

■about 50% of calcium in blood is bound to albumin and is inactive;
the other 50%, called free or ionized calcium, is metabolically
active. Total calcium is a measurement of both bound and free
calcium..