Determine whether each of your 4 suspect drivers are ‘guilty’ or ‘not guilty’ of driving

over the legal BAC of 0.05%(w/v). How confident are you in your estimate of each
driver’s BAC?

Driver A, C and D are not guilty. Driver B is guilty.

The experiment design ensured the BAC of each of the drivers was obtained with great
confidence. The absorbance of all 4 of the blood samples lies between the recommended 0.1-1
range, which increased the accuracy of the final concentrations obtained. Moreso, the
experimental design took advantage of the 0x speed function of the timer by activating before
adding the enzyme to all standard and blood sample dilutions. The timer was then adjusted to a
normal speed to ensure the reaction time was constant for all the standard and blood sample
dilutions. Moreso, the absorbance was collected every 10 minutes until 30 minutes - 3
absorbance readings were recorded for each reaction to ensure it had reached completion.

However, driver C’s blood BAC of 0.045 %(w/v) lies close to the threshold. If there is any human
error with the collected data, the BAC may lie above the threshold. Therefore, it cannot be
confirmed that the driver has not exceeded the legal 0.05% (w/v) threshold with high
confidence.

Why might you get very different estimates for an unknown? What might have
contributed to this variation? How would you decide which estimate (which of
your three replicate samples) was most accurate? (1 mark)

The reliability of the standard curve’s line of best fit is reduced because it does not go through
all 6 experimentally obtained data points. Thus, the line of best fit is fairly an estimation of the
relationship between absorbance and concentration. This estimation contributes to the variation
in BAC concentrations obtained in the three replicates of each blood sample. The replicate
closest to the middle 0.5 absorbance can be used to decide the most accurate replicate sample.

1. What would happen if you made a standard curve using 0 to 30 μL

instead? You can test this within the ELMA Data Generator to help
answer this question if you like. (1 mark)
The standard curve will eventually reach a constant absorbance value at some point before 30
μL.
2. What might happen if you added vodka (37% w/v) to your assay plate
instead of one of your blood samples? What additional steps would you
need to include to be able to measure the concentration? You can test
this in the ELMA Data Generator to help answer this question if you like.
(1 mark)
 3. What additional steps could be taken to give you more confidence in
your estimates? You can test this to help answer this question if you
like. (1 mark)
- The average concentration was calculated for each blood sample to increase accuracy.
However, the whole experiment could be repeated at least 3 times to increase the reliability of
the experiment.
- The current experiment diluted each blood sample three times to calculate a final average
concentration. In order to increase the accuracy of the concentrations, each blood sample
should be diluted at least 5-6 times.

4. You notice that one of your patient samples has turned a red colour due
to haemolysis (lysis of red blood cells). What effect do you think this
will have on your assay? What steps could you take to correct this? (2
bonus marks).
Haemolyisis can disrupt the reliability and accuracy of the assay. The lysis of blood involves the
release of intracellular substances from red blood cells which can impact the absorbance
reading of the analysis. Ultimately, changing the actual concentration of the driver’s blood
sample.

In order to correct the impact of haemolysis, the assay should be repeated with a fresh new
sample to ensure accuracy. To detect any possible haemolysis, controls and markers should be
used in this new blood sample. Any damaged cells in the supernatant should be separated
through a centrifuge. In order to account for any disruptions from released intracellular
components, the assay reagents or protocol should be adjusted.