DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404
Title:
Effects of specimen handling on analytes

Objectives:
To determine the effect of several preanalytical factors on the level of
analytes

Principle:
Results from the laboratory test are used for detection and diagnose
of disease. Its also can be use to monitor the progress or its response to
treatment and to screen for disease in otherwise healthy individuals. If the
result is abnormal, it shows a disease state in patient
However, many factors also can affect the composition of body
fluids. These factors may be analytical or preanalytical. Thus, wherever
possible, both preanalytical and analytical variability should be controlled
so that correct interpretation of the result can be made In this experiment
we are going to see some effect of preanalytical factors on the level of
glucose and potassium in serum

Procedure:
1. 10 ml blood was drawn from each student
2. Blood was divided into 5 tubes
(a) Room temperature. The blood was centrifuge at 3000rpm for 15
minutes. The plasma was taken for the result of glucose and
potassium concentration
(b) Lysed, room temperature. The blood was centrifuge at 3000rpm for
15 minutes. The plasma was taken for the result of glucose and
potassium concentration
(c) At room temperature and was incubated for over night. The blood
was centrifuge at 3000rpm for 15 minutes. The plasma was taken
for the result of glucose and potassium concentration
(d) 40C and incubate for over night. The blood was centrifuge at
3000rpm for 15 minutes. The plasma was taken for the result of
glucose and potassium concentration
(e) -200C and incubate for over night. The blood was centrifuge at
3000rpm for 15 minutes. The plasma was taken for the result of
glucose and potassium concentration

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 3. The serum was separated on the specified day
4. The glucose and potassium level in tube (a) and (b) was measured using a
reflotron analyzer
5. The analyte level in tube (c), (d), and (e) was measured on the next day
6. The result and observe of any changes in the reading was recorded.
Result:
Reference of glucose and potassium level:
Glucose: 4.22 – 6.11 mmol/L
Potassium: 3.5 – 5.1 mmol/L
Tubes Glucose level Potassium level
(a) 5.29 mmol/L 4.23 mmol/L
(b) 5.87 mmol/L 4.40 mmol/L
(c) <0.555 mmol/L 4.45 mmol/L
(d) 3.15 mmol/L 9.19 mmol/L
(e) 8.08 mmol/L >12.0 mmol/L

Discussion:
Calculations of K+ concentration in blood
 The principle of the reaction involve the diffusion of potassium from the
aqueous phase into the organic phase where it forms a complex with
valinomycin
 In order to balance the charge, a pH indicator was dissolved in the organic
phase. It gives off a proton and forms a colored anion.
 An optimum change of the reflectance was achieved by addition of a
strong acid, which forms a colorless anion that compete with the indicator.

Calculations of glucose concentration in blood

 The reaction below showed the chemical reaction involves in this process
Glucose + O2 Glucose oxidase
δ-D-gluconolactone + H2O2
H2O2 + indicator Peroxidase
dye + H2O

The result in tube A:

 Tube A act as a reference or control of this experiment
 The result from the experiment shows that the blood glucose and
potassium level is in a normal state

The result in tube B:

 In tube B the red blood cell are lysed. As the result potassium and glucose
from the intracellular was released into the extracellular fluid, in this case,
serum
 The release of this amount cause a little increase in the concentration of
glucose and potassium in the serum

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  However the glucose and potassium concentration in this tube is still
within the range of the normal potassium and glucose level.

The result in tube C:

 In tube C, the blood sample was leave for overnight at room temperature
 This process allowed the cells to respirate and undergo metabolic
activities
 When the amount of an analyte (glucose) was lost due to respirate and
metabolic activity (glycolysis process), there’s only little amount of
glucose left.
 As the result the glucose concentration decrease
 Meanwhile for potassium, there is a fact that the potassium level would
increase about 2mmol/L in every 5 hours. However the result only
showed a little increase due to some error that occur during blood intake
procedure

The result in tube D:

 Usually blood start to haemolyse in low temperature.
 Glycolitic process occur and it cause the consumption of glucose therefore
the glucose level decrease.
 However, the potassium level still increase about 2mmol/L in every 5
hours
 We can see in the result of this experiment, that shows the slightly
increase in the concentration of potassium after incubate for overnight in
40C

The result in tube E:

 When the blood was freeze at -200C, it will induce haemolysis, much
heavier than letting it cool at 40C.
 In this case, the glycolitic activity cannot occur due to low temperature.
 No enzyme reaction, so glucose was used.
 However the red blood cell still lyse and the glucose that came out from
red blood cell cause the increase in the glucose concentration
 The potassium level is still increase.
Reason for error result:
 The wrong procedure in vein puncture that can cause the haemolysis of
the blood
 Forcing the blood into the tube also can cause haemolysis
 The vigorous shaking and let the blood sample for a long time before
measuring the analyte concentration also an cause error in the result

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Conclusion:
 Usually normal human body contains 50mmol/L potassium per kg body
weight.
 It is important to maintain the concentration gradient between
extracellular and intracellular in order to maintain the membrane
potential and excitability.
 Glucose plays an important role as body energy source. It is stored as
glycogen and was broken down by glycolysis process.
 The normal glucose level in body should be 4.22 – 6.11 mmol/L
 The normal potassium level in body should be 3.5 – 5.1 mmol/L
 The result can be affected by various ways such as an error in blood intake
(vein puncture) and other methods.