Chemistry Equations

Question 1

Magnesium (Atellica)

Answer 1

Mg2+ + xylidyl blue
-> (OH-)
Xylidyl blue Mg complex

Increase in absorbance at 505/694

Question 2

Calcium (Atellica)

Answer 2

Ca2+ + Arsenazo III
-> (pH 5.9)
Ca-Arsenazo III complex

Increase in absorbance at 658/694

Question 3

Total protein (Atellica)

Answer 3

Peptide bonds react with cupric sulfate (biuret reagent) in an alkaline environment - “Weichselbaum method”
Protein + CuSO4 –> (OH-) Cuproproteinate complex
Measured at 545nm

Question 4

Indirect (total) bilirubin and direct bilirubin - method

Answer 4

Indirect:
All bilirubin is made water soluble first by accelerator (caffeine benzoate in Jendrassik Grof method (reference method)
Addition on alkaline tartrate to shift absorption ?avoid haemolysis interference)
Bilirubin + vanadate (detergent + pH 2.9) –> Biliverdin
Decrease in absorbance (of bilirubin) at 451/545nm
Endpoint reaction

Direct:
Conjugated bilirubin + vanadate (pH3 + detergent) –> bilverdin
Decrease in absorbance at 451/545

Question 5

What is delta-bilirubin, and which analyser can measure it?

Answer 5

conjugated bilirubin + albumin
determined on Vitros analyser

Question 6

Biilirubin interferences

Answer 6

degraded by direct light
heparin effects
haemolysis - particularly conjugated on Abbott archi

Question 7

Advantages of the Vitros dry method for bilirubin

Answer 7

Masking layer prevents proteins incl delta bili and hb from being included in measurement - delta-bilirubin measurement can be determined and excluded from the measurement of conjugated bilirubin in neonates - all neonatal bilrubins are done by Vitros at PathWest.
Less affected by haemolysis

Question 8

CK (Atellica equation)

Answer 8

Modified procedure of Szasz
Concentration of NADPH measured by increase in absorbance at 340/596nm.

Question 9

LDH (Atellica equation)

Answer 9

Amount of NADH produced measured by increase in absorbance at 340/410 nm

Question 10

Amylase method/equation

Answer 10

Released p-nitrophenol is measured at 410/694nm

Question 11

Lipase method

Answer 11

Production of methylresorufin is measured at 571/694nm

Question 12

ALT method

Answer 12

Decrease in absorbance of NADH is measured at 340/410nm

Question 13

AST method

Answer 13

Decrease in absorbance measured at 340/410nm

Question 14

ALP method

Answer 14

Measured using a bichromatic rate technique. Change in absorbance of p-nitrophenol at 410nm directly proportional to ALP.

Question 15

Creatinine method (Atellica)

Answer 15

Modified Jaffe reaction: Creatinine reacts with alkaline picrate to form red-coloured creatinine picrate, detected at 505/571nm. Modified to include rate blanking (to minimise bilirubin interference) and intercept correction (as non-specific protein interactions with the reagent produce a positive bias).

Question 16

Phosphate method (Atellica)

Answer 16

Pi + ammonium molybdate (H2SO4) -> phosphomolybdate detected by spectrophotometry as an endpoint reaction at 340nm/658nm

Question 17

Urea (Atellica)

Answer 17

Urea is hydrolysed by urease to ammonia and carbon dioxide. In the second step, glutamate dehydrogenase catalyses to conversion of ammonia and alpha-ketoglutarate to glutamate, oxidising NADH to NAD. The decrease in NADH concentration is measured as an inverse rate reaction at 340/410nm.

Question 18

Ammonia (Beckman AU680)

Answer 18

Glutamate dehydrogenase catalyses the conversion of ammonia and alpha-ketoglutarate to glutamate, oxidising NADH to NAD in the process. Decrease in NADH is measured at 340/410nm.
LDH is added in excess to rapidly reduce endogenous pyruvate so that it does not interfere with the assay system

Question 19

Uric acid method (Atellica)

Answer 19

Uric acid + water + O2 catalysed by uricase to allantoin, CO2 and hydrogen peroxide
Hydrogen peroxide enters a Trinder-like reaction to form a quinone diimine dye the absorbance of which is measured by spectrophotometry and is directly proportional to the uric acid concentration.

Question 20

Glucose method (Atellica)

Answer 20

Glucose + ATP catalysed by hexokinase to glucose 6 phosphate
Glucose 6 phosphate + NAD catalysed by G6PD to 6 phosphogluconate + NADH
Absorbance of NADH measured by spectrophotometry proportional to concentration of glucose.