Analysis and Diagnosis of a clinical case using clinical, biochemical and analytical methods

1. Introduction
The aim of the investigation is to analyse this
clinical case using clinical, biochemical and
analytical methods to identify and highlight
abnormalities that may be found upon testing the
patient’s serum and urine samples, to ultimately
inform the diagnosis of the conditions. The tests
will be carried out efficiently and accurately but
also as ethical as possible meaning the patient’s
samples were not used for unnecessary testing.
Patient Background:
A 9 year old male. Reports of suffering with
drowsiness, tiredness, frequently drinking and
passing more urine than usual. Furthermore
recent vomiting and loss of consciousness in
hospital. Respiration is deep, he has cold
extremities and blood pressure is low. Parents
are concerned as they have identified an
unknown powder in the boys room.
Figure 2. Results of Glucose Oxidase assay
presenting concentration of glucose (mmol/g)
measuring from 0-20mmol/g at 570nm.
Results and Discussion
Spectrophotometric Glucose Oxidase assay
Glucose Oxidase assay was used to determine
the concentration of glucose in the patient sample
by using the specificity of enzyme reactions which
generated coloured molecules which were
measured by spectrophotometry. Enzyme
glucose oxidase was used to oxidise glucose to
gluconic acid and hydrogen peroxide. Hydrogen
peroxide is further used to oxidise ABTS to a
coloured product as peroxidase catalyses the
reaction. This amount of coloured product is
measured using spectrophotometry and is
proportional to the original concentration of
glucose in the patient sample.
Department of Biosciences
Authors: Thomas Lewis
Department of Biosciences and Chemistry, Faculty of Health and Wellbeing, Sheffield
Hallam University, Sheffield S1 1WB, United Kingdom
Analysis & Diagnosis of a Clinical Case using
Clinical, Biochemical & Analytical Methods
Techniques
• Dipstick Urinalysis
• TLC for drugs
• Spectrophotometric Glucose Oxidase assay
• Serum Creatinine Assay
• Flame Emission Spectroscopy using Flame
Photometers
1) Glucose + O2 + H2O (glucose oxidase) 
Gluconate + H2O2
2) H2O2 +ABTS (peroxidase)  coloured
complex + H2O
Figure 1. The reaction of oxidation of glucose to
gluconic acid & hydrogen peroxide followed by
reaction of oxidation of ABTS to coloured product
by hydrogren peroxide.
Concentration of
Glucose (mmol/g)
Mean absorbance
@ 570nm
0 0
0.2 0.015
0.5 0.055
1 0.098
2 0.204
4 0.416
6 0.636
8 0.875
10 1.122
12.5 1.416
15 1.667
20 2.136
EQA A 1.319
EQA B 1.768
EQA D 1.268
High QC 0.687
Low QC 0.997
Patient sample 1.136
Glucose oxidase standards ranging from 0-
20mmol/g were prepared from a 20mM stock
solution. With the addition of glucose oxidase
reagent to each standard and also EQA, QC and
patient samples. After incubation for 25 minutes
the samples were read using spectrophotometer
at 570nm and results collected.

2. Materials and Methods
Dipstick Urinalysis
Basic urine examination to determine glucose,
protein (albumin), ketones concentration and pH
of urine by dipping test strip into patient urine
sample. This technique works as the acid-base
indicator changes colour in the presence of i.e.
protein (albumin) as protons are removed from
the indicator and bind to albumin binding sites
(Bartos, et al. 2016).
TLC for drugs
Simple analysis technique used to determine the
identity of unknown powder content found in
Patient A’s bedroom. Standards of analgesics
were run to compare the unknown to. Caffeine
standard was also run. Solution was extracted
from the unknown powder by dissolving in
ethanol. The standards and unknown were
spotted onto TLC plate which was then placed in
TLC developing solvent and left to run until
solvent had risen just above the top of the plate.
Analysis was then undertook by calculating Rf
value to determine identity of unknown.
Flame Emission Spectroscopy using Flame
Photometers
Bioanalytical technique using Flame Photometers
(FP) to determine levels of serum electrolytes
Na+ and K+ in patient sample. FP uses the
principle of a simple flame test by introducing the
sample to the flame. The electrons become
excited but then lose energy by emitting light of a
specific wavelength. This is measured allowing
the concentration of the electrolytes to be
estimated. Sets of standards were produced
using stock sodium and potassium solutions. The
standards were then aspirated followed by QC
samples and the patient sample. A calibration
graph was plotted for each electrolyte and the
patient sample read off each to determine
electrolyte concentration.
Conclusions
Dipstick urinalysis shows abnormally high glucose
concentration in patient urine, supported by
further analysis by glucose oxidase assay.
Serum creatinine analysis proved to be
inconclusive however high glucose concentration
and low potassium and sodium levels are traits
associated with diabetes or renal failure.
References
Vladimir Bartos et al. (2016). Clinical
Biochemistry (1st ed). ISBN 978-80-246-3497-5
(pdf)
Additional Results
Dipstick Urinalysis
This method of determination is most sensitive to
protein (albumin) and the result of protein in
Patient A urine sample was negative. Dipstick
urinalysis also determined an abnormality in
slightly raised ketones concentration at 15mg/dL,
which can cause low K+ levels as found below
from Flame Emission Spectroscopy. However pH
of patient serum sample was within healthy range
and no concern.
TLC for drugs
This method determined the putative identity of
the unknown powder as paracetamol.
Flame Emission Spectroscopy using Flame
Photometers
This method determined the concentration of Na+
in the patient sample to be 40 mmol L-1 and the
concentration of K+ to be 0.9mmol L-1. These
results are abnormally very low.
Serum Creatinine Assay
Calculation to determine creatinine concentration
proved to be inconclusive therefore if there was
more time this assay should be conducted again
to conclude the clinical investigation.
-0.5
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25
Absorbanceat570nm
Concentration (mmol L-1)
Determination of glucose
concentration in biological sample
using spectroscopy.
Figure 3. Calibration graph of patient sample
using glucose oxidase assay to determine
glucose concentration.
Glucose concentration from original patient
sample was calculated = 53 mmol L-1. This result
certainly is abnormal as it is very high above the
normal reference range of a healthy individual.
Serum Creatinine Assay
Standards and patient samples were prepared
and assayed using 96-well plate protocol before
being loaded with working reagent. Plate was
incubated for 1 minute then the absorbance
measured at 492nm by plate reader. After a
further 4 mins incubation the absorbance was
measured again.
Sample Abs @
492nm 1min
Abs @
492nm 5min
1 0.044 0.046
2 0.045 0.046
3 0.043 0.045
Mean 0.044 0.0456
Patient 0.0463 0.0463
Control 0.043 0.0443
Figure 4. Results of Serum Creatinine assay
presenting absorbance of samples at 492nm at
1 minute and 5 minutes.