DIAGNOSTIC IN CLINICAL CHEMISTRY

I
MKEB 2404

TITLE:
• Effect of specimen handling on analytes.
DATE:
• 10 JANUARI 2007
AIMS AND OBJECTIVES:
• To determine the effect of several preanalytical factors on the levels of analytes.
PRINCIPLE:
• Results of laboratory tests are used by physicians to diagnose disease, to
monitor its progress or its response to treatment and to screen for disease in
otherwise healthy individuals.
• Abnormal test result will indicate a disease state in the patient.
• However, many factors aside from disease 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.
• This practical aims to demonstrate the effect of several preanalytical factors on
the level of glucose and potassium in serum.
PROCEDURE:
1. Draw 10ml of blood from each student.
2. Divide blood into 5 tubes. Label the tubes as:
(a) Room temperature
(b) Lysed, room temperature
(c) Room temperature, overnight
(d) 4°C, overnight, and
(e) -20°C, overnight.
3. Separate the serum on the specified day.

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4. Measure the glucose and potassium levels in tube (a) and (b) by using a
reflotron analyser.
5. On the next day, measure the analyte levels in tube (c), (d) and (e).
6. Record the results and observe any changes in the reading.

INTENDED USE
• The quantitative test for determination of potassium in serum with Reflotron.
• The quantitative test for determination of glucose in serum with Reflotron.
CLINICAL ASPECTS
• POTASSIUM TEST
The human body contains about 50 mmol potassium per kg body weight. About
90 % of this is contained in the cells, only 2 % in the extracellular space. This
concentration gradient between the intracellular and extracellular space is essential in
order to maintain the membrane potential and the excitability of the cells.
The daily requirement of 30 - 50 mmol potassium is satisfied by dietary intake,
absorption takes place mainly in the small intestine. About 90 % of potassium excretion
takes place via the kidneys and about 10 % with the faeces.
Disturbances of potassium homeostasis, in whichever direction, are relatively
poorly compensated and can rapidly lead to life-threatening situations particularly
with regard to cardiac function.
• GLUCOSE TEST
The body obtains glucose from glucose-supplying carbohydrates. It is the main
monosaccharide in the blood with a postprandial concentration of 5 mmol glucose per
litre and is an indispensable energy-delivering substrate for cellular functions.
Glucose is broken down by glycolysis. Determination of glucose is used for the
diagnosis and monitoring of disorders of carbohydrate metabolism such as diabetes
mellitus, idiopathic hypoglycaemia and in pancreatic islet cell tumours.

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TEST PRINCIPLE:
• POTASSIUM
After application to the test strip, the sample flows into the reaction zone. In a
reagent film consisting of two phases the potassium diffuses from the aqueous into the
organic phase and is complexed by valinomycin. To balance the charge, a pH indicator in
the organic phase gives of f a proton, yielding an anion.
A strong acid competing, with the indicator and forming a colorless anion
permits an optimal change in reflectance.

• K+ + Valinomycin + Indicator [Valinomycin-K]+ [Ind]-+H+

• K+ + Valinomycin + Acid [Valinomycin-K]+ [A]-+H+

• GLUCOSE TEST
After application to the test strip, the sample flows into the reaction zone, in the
case of blood after separation of the erythrocytes from the plasma. D-glucose is oxidized
to δ-D-gluconolactone by atmospheric oxygen in the presence of glucose oxidase
(GOD). The resulting hydrogen peroxjde oxidizes an indicator in the presence of
peroxidase (POD). The dye formed in this manner is proportional to the glucose
concentration of the sample:
• Glucose + 02 δ-D-gluconolactone + H202
• H202 + Indicator Dye + H20

a) Unwrap the strip, taking care not to bend it and do not touch the black barcode
on the strip. Using the Reflotron pipette, draw sample serum into apply it onto
the centre of the yellow application zone. Be careful not to touch the application
zone with the pipette tip.
b) The required volume of serum is 30 μl.
c) Then, place the test strip on to the machine and slide forward horizontally until
it locks into place. Close the sliding cover.
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REFERENCES RANGES:
• POTASSIUM
Serum: 3.6 - 5.0 mmol/l
• GLUCOSE
Fasting glucose adults: 3.3 - 6.05 mmol/l.

SAMPLE COLLECTION:
• Serum or heparinized plasma obtained with standard sample collection tubes.

RESULTS:
TUBE GLUCOSE LEVEL POTASSIUM LEVEL
A 5.72 mmol/L 3.89 mmol/L
B 4.51 mmol/L 5.36 mmol/L
C < 0.555 mmol/L 3.47 mmol/L
D 3.36 mmol/L 8.46 mmol/L
E 7.24 mmol/L >12.0 mmol/L

DISCUSION:
• TUBE A
○ On the Tube A, show that the serum sample of the both test are in the
normal range.
○ Where, this sample taken as control for this experiment.
• TUBE B
○ In the tube B, the samples are hemolysed by using the high vortex mixer.
○ Where after the sample is spin out, the serum looked slightly reddish.
○ So, as a result:
 Glucose :
➢ The value is decrease than in the Tube A.
➢ Interpretation:
 This is because of the activity process of glycolysis.
 Where, the sample is mixed up with hemolysed cell
that increase the activity process.
 Potassium :
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 ➢ The value is increase than in the Tube A.
➢ Interpretation:
 This is because of the hemolysed sample that affects
the value result.
2. TUBE C
○ On the tube C, the sample is stored overnight at room temperature.
○ After, the sample is analyzed, the result are shown;
 Glucose :
➢ The glucose value is become more decrease than other
tubes before.
➢ Interpretation:
 This is because of the more activity process of
glycolysis happen, when it stored at room
temperature.

 In the addition, the sample also not separated from

the cell, which will make more increasing the
process of the glycolysis.
 Potassium :
➢ They are nothing much change of the potassium value than
other tubes before.
➢ Interpretation:
 This is because the samples that leave in the room
temperature for overnight not affect the value of the
potassium.
 But, if the sample is stored more than 3 days, the
potassium value will be change.
3. TUBE D
○ In the Tube D, the sample is stored for overnight at 4’C temperature.
○ As a result;
 Glucose :
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 ➢ The glucose value is normal.
➢ Interpretation:
 This is because of the temperature factor, where it
will hold or slow down the glycolysis process than
happening.
 Potassium :
➢ They have gradual change of the potassium value than
other tubes before.
➢ Interpretation:
 This is because the samples that stored in the high
temperature without separating the serum will be
increasing the hemolysed.
 Therefore, the value of the potassium will also
increase.

4. TUBE E
○ In the Tube D, the sample is stored for overnight at -20’C temperature.
○ As a result;
 Glucose :
➢ The glucose value is higher than other tubes before.
➢ Interpretation:
 This is because of the temperature factor, where the
glycolysis process is fully stopped.
 And, as addition the serum become more hemolysed
with the increasing of the temperature.
 Potassium :
➢ They are also gradual changes to potassium value.
➢ Where the machine can’t give the true result because of the
out of range of measuring.
➢ Interpretation:

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  This is because of the temperatures factor also,
where the sample is stored higher than the tube D.
 So, therefore the value of the potassium value for
tube E becomes more increase.
PRECAUTION OF THE TEST:
1. The blood sample must be clotted first before spinning, this is because to
prevent the serum clotting together with the cell. By leave it the blood sample
around 15 minutes.
2. For potassium analysis, the tubes are used is not contain any anticoagulant.

CONCLUSION:
1. Conclusion for this experiment, which the normal value for fasting blood
glucose, is 4.22 - 6.11 mmol/L.
2. And, the normal value for the potassium is 3.5 – 5.1 mmol/L.
3. Table below shown factor that affecting the serum value of the glucose and
potassium.
TEST FACTOR VALUE
1. MEALS INCREASE
2. SMOKING INCREASE
3. CAFFEINE INCREASE

4. SMOKING INCREASE

GLUCOSE 5. CHRONIC DECREASE

CONSUMPTION OF
ALCOHOL
6. INTRAVENOUS DECREASE

CONTAMINATION
POTASSIUM 1. PROLONGED INCREASE
TOURNIQUET
2. HEMOLYSIS INCREASE

3. USING EDTA TUBES INCREASE

INCREASE
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4.ANTICOAGULANT DECREASE
5. DIURETICS DECREASE
6. OPENING AND
CLOSING OF HAND
BEFORE TAKEN
BLOOD

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