1 INTRODUCTION TO LABORATORY INVESTIGATION
INVESTIGATION OF GLUCOSE METABOLISM
Topics to be reviewed
Blood and its components. Blood plasma and serum. Anticoagulants. Metabolism of glucose.
Glycemia and its regulation. Disorders of glucose metabolism. Glucosuria.
Pre-lab questions
1. Which factors can induce hemolysis during blood collection and blood processing?
2. Give examples of disinfectants used during blood collection.
3. Why is NaF added to collected blood before glucose determination?
4. Draw structural formulas and names of ketone bodies.
1.1 Importance of laboratory biochemical investigations
For accurate appraisal of health state many information should be acquired. Results of laboratory
examinations are considered as the source of valid information needed for the assessment of health
state and the reflection of metabolism changes in body. According to World Health Organisation
(WHO) laboratory examinations provides about 80 % information leading to the assessment of correct
diagnosis.
The physician can use a variety of laboratory methods during a different phases of diagnostic and
therapeutic process. However it is necessary to indicate rationally and to use effectively these methods
and then to evaluate and correctly interpret obtained results There are known basic, specialized and
highly specialized laboratory investigation. According to rate of carrying out we distinguish routine
and urgent laboratory investigation. A small number of simple, mostly qualitative tests with direct
informative results take place in the doctor’s surgery or at the patient’s bed (bed-side diagnostics,
point-of-care testing, near-patient testing).
1.1.1 Handling biological material, blood collection
The largest and most important part of the clinical-biochemical examinations concerns blood, blood
serum and plasma analyses. Another frequently analysed material is urine . Analyses of the other body
fluids (gastric juice, duodenal juice, amniotic fluid, cerebrospinal fluid, saliva, transpiration, etc.) are
demanded only in a chosen number of patients, their frequency is much lower, and they are often
carried out in the special laboratories.
Blood is most frequently analysed biological material. Blood is easily accessible material and its
composition reflects the range of biochemical processes blood is a good indicator of the physiological
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conditions throughout the body. Blood for analysis may be taken from veins, arteries, or capillaries.
Venous blood is usually the specimen of choice, for some analysis and especially in young children is
taken capillary blood. The arterial puncture is used only exceptionally, especially for the blood gas
analysis.
When blood is drawn without the addition of an anticoagulant and allowed to stand, it clots after
several minutes, because soluble fibrinogen is transformed to the insoluble network of fibrin by the
clotting mechanism. Serum is excluded from the blood clot after some time and is obtained from
clotted blood by centrifugation. Sufficient time is needed for complete blood clotting (at room
temperature at least 15–30 min).
Non-clotted blood is required for some clinical-biochemical examinations. Blood is taken to the tubes
with the addition of the anticoagulants (sodium oxalate, sodium citrate, EDTA Na2, heparin).
Centrifugation of non-clotted blood produces plasma. Blood may be centrifuged immediately after the
collection. Plasma or serum should be separated from blood cells or coagulum within 2 hours at the
latest (or 1 hour for the determination of potassium ions).
1.1.1.1 Venous blood collection
With regard to the method and equipment used for the venipuncture are distinguished two ways of
collection
• Open collection system - phlebotomist is in direct contact with the biological material
– blood is drawn through needle directly into a test tube
– blood is drawn into a disposable syringe and by back sticking into a test tube.
• Closed collection system – the sample handling is carried out after the collection directly in the
collection syringe or test tube. Thus, the laboratory technician is protected against the contamination
by the patient’s blood, the biological material is protected against the external contamination, and also
against breaking during transport and centrifugation. The individual syringes/evacuated test tubes are
marked by colour according to the additive used (clotting accelerators, separation gel, heparin, EDTA,
...). The separation gel enables proper separation of serum from the blood coagulum after
centrifugation (creation of interlayer). The collection syringe/test tube can be placed directly into the
analyzer. The used material is easily burnt down.
The collection is carried out:
- into the closed syringe – the blood is drawn out with the help of a piston (Fig. 1-1a) or with
immediately before the collection prepared vacuum (pulling the plunger into the base of syringe and
locking it into place, Fig. 1-1b). After the collection, the piston is broken off and the syringe is
transformed into the closed test tube. The method of collection is adapted to patients with problematic
veins. Using piston we collect the blood samples in patients with thin veins. Vacuum is used in case
of strong veins
- into the evacuated test tube (Fig. 1-1c)
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Evacuated
test tube
Holder
Double spiked
needle
Haemostatic ventil
Fig. 1-1a Fig. 1-1b Fig. 1-1c
Specimen collection general instruction
• 10–12 hours before the blood withdrawal patients are not allowed to eat; they have to exclude fat
food and alcohol from their diet.
• In the morning before the blood withdrawal, patient can drink about ¼ L of plain water or bland tea.
• Before the blood withdrawal, the patient should be asking for any allergies to antiseptics, adhesives.
• Standard posture of patient at the blood withdrawal is sitting posture.
• Pumping of the fist before venipuncture should be avoided (results of many tests can be affected).
• The site of injection must be disinfected (Jodonal B, Persteril, Jodisol, Ajatin).
• If the veins are visible, the use of a tourniquet should be minimized.
• If the tourniquet is used (not more than 1 min), it should be released before withdrawal of blood
begins.
• Press down on the gauze once the needle is out of the arm (to avoid formation of a hematoma). The
gauze should be moisturized in disinfectants.
• If the non-clotted blood is drawn out, it is necessary to mix blood immediately after collection by
rotating the tube at least five times.
1.1.1.2 Capillary blood collection
Capillary blood is used in case only a small amount of the sample is needed. General instructions for
specimen collection:
• Warm the site of injection (tip of the finger, an earlobe, the heel or big toe of an infant), ensure good
blood supply. Avoid massaging the site.
• The injection site must be disinfected.
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• Puncture should be directed onto the outer and upper region of the fingertip, halfway between the
centre of the finger pad and the edge of the fingernail.
• After puncture with the lancet, the first drop should be wiped off (dilution of blood with the tissue
fluid). Gently apply intermittent pressure to the surrounding tissue. Avoid excessive squeezing or
"milking" of the puncture site.
• After puncture with the lancet, the first drop should be wiped off (dilution of blood with the tissue
fluid). Gently apply intermittent pressure to the surrounding tissue. Avoid excessive squeezing or
"milking" of the puncture site.
• Blood is usually collected into the special capillary blood test tubes or into the small plastic or glass
test tubes. For strip tests, the drop of blood is applied directly on the paper.
• Following collection press clean gauze moisturized in disinfenctants on the puncture side.
► See video recording and acquaint with the blood withdrawal ways.
► Draw chemical formulas of common anticoagulants and explain how they act.
► Describe the appearance of blood before and after being centrifuged
► Give the main differences in composition of blood plasma and serum.
► What does it mean, when we describe plasma/serum as a) hemolytic; b) icteric; c) chylose?
1.2 Investigation of glucose metabolism
Glucose concentration in blood, plasma, or serum is the basic biochemical value of saccharide
metabolism. The most frequent type of disorder of saccharide metabolism is diabetes mellitus (DM).
This metabolic disease can occur, if fasting blood glucose is above 7 mmol/L. To confirm the
diagnosis, if not determined from glycemia1
, the glucose tolerance test is used. We use other
biochemical examinations in order to monitor the course of diabetes and to control the therapy: the
quantitative determination of glucose in urine, glycated hemoglobin in blood, the detection of
microalbuminuria and ketone bodies in urine.
Diabetes mellitus is a condition characterised by absolute or relative insulin deficiency. DM is divided
into several forms and stages, which must be distinguished from prognostic as well as therapeutic
reasons. According to etiology, we recognize DM of the 1st
type, DM of the 2nd
type, gestational
diabetes, and other specific forms of diabetes.
Diabetes mellitus of the 1st
type is a polygenic autoimmune disease. It is a less frequent type of
diabetes. Genetic predisposition combined with external factors (such as viral infection, toxins, stress)
can induce pre-diabetic phase of the disease, which can last several years. In this time, the β-cells of
the Langerhans’ isles are slowly destroyed by activated T-lymphocytes and cytokines, a condition
expressed as insulitis (the lymphocytic infiltration of the cells of the isles, inflammation). Insulitis
gradually reduces the number of functional β-cells, which results in the disruption of synthesis and
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The more accurate term for the description of the blood level of glucose is glucosemia. However, the literature commonly
relies on the term glycemia.
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secretion of insulin. Diabetes is clinically manifested, when 60–70 % of pancreatic β-cells are
destroyed by the autoimmune inflammation.
Diabetes mellitus of the 2nd
type results from insulin resistance and/or relative insulin deficiency
(abnormal insulin, insulin antibodies). It is the most frequent type of DM. Insulin resistance can be the
result of the decreased number of plasmatic membrane receptors on the target cells or the consequence
of the post receptor blockade of the intracellular metabolism of glucose (decreased number of
receptors or decreased affinity; decreased activity of the pyruvate dehydrogenase complex; abnormal
signal transfer or abnormal phosphorylation reaction for the excessive production of TNFα). The level
of insulin insufficiency reflects the gradual loss of the potency of β-cells to respond to glucose.
1.2.1 Enzymatic determination of plasma glucose
Glucose oxidase (GOD) catalyses the oxidation of β-D-glucopyranose by air oxygen to δ-lactone of
gluconic acid and hydrogen peroxide:
β-D-Glucopyranose + O2 D-Glucono-1,5-lactone + H2O2GOD
In the presence of peroxidase (POD), hydrogen peroxide then reacts with the chromogenous reagent
(H2A) to form the red coloured product (A):
H2O2 + H2A 2 H2O + APOD
Maintaining the prescribed reaction conditions, the amount of this product is proportional to the
concentration of glucose. This reaction is sensitive and fast. The resulting colour is measured after
reaching the final equilibrium (end-point method). It can be also measured continuously during the
first few minutes after initiating of the reaction (kinetic method).
It is not necessary to adjust serum or plasma samples, if they are separated from whole blood
immediately after blood collection. Otherwise, blood samples should be stabilized: the simplest way is
blood cooling or adding of mannose (it is alternative substrate for hexokinase and acts immediately) or
NaF (it inhibits glycolysis in red blood cells after entering into cell by diffusion, i.e. after about 2
hours).
Materials: Kit Glu GOD (Erba Lachema*): Reagent-glucose (containing 3-methylphenol 11 mmol/l; 4-aminophenazone
0,77 mol/l; glucose oxidase ≥ 300 μkat/l; peroxidase 18,3 μkat/l), calibrator-glucose (concentration is given
on the label of vial). Specimen of blood serum. Pipettors 20 μl and 2 ml, water bath at 37 °C.
Spectrophotometer Spekol 1300 and software WinAspect or Helios Delta and software VisionLite Fixed.
*Alternatively, the kit of Roche-Diagnostics, Human or BioVendor can be used; the composition of reagents
may be then different.
Procedure
Deproteinization is not essential in the case of non-hemolytic blood serum or plasma. Pipette the
components into clean test tubes according to the following table:
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Reagens (µl) Blank Test Standard
Reagent-glucose 1 000 1 000 1 000
Demi-water 20 - Serum
- 20 Calibrator-glucose
- - 20
Shake all the test tubes vigorously (important for saturating with oxygen).
Incubate all tubes for 30 min at room temperature (or 15 min at 37°C)
Avoid exposure to direct light.
Read the absorbances*
of sample (Ax) and standard (ASTD) at 495 nm against the blank within 40 min.
*Measure absorbances according to the enclosed instructions to the spectrophotometers and use relevant software.
Calculation of the concentration of serum glucose: STD
STD
x
x c
A
c ×=
A
where cSTD is the concentration of glucose in calibrator (it is given on the label of the vial).
Evaluation
Reference values of plasma glucose concentration at fasting state in healthy adults (FPG-fasting
plasma glucose): 3,9 – 5,6 mmol/l
For precise determination of glycemia, we should be aware of the fact that the reference levels differ
according to the type of processed material. The levels of glucose in plasma are approximately about
10-15% greater than in whole blood. There are also differences between capillary and venous blood.
The levels in capillary blood are greater than in the venous blood, at fasting about 5% and postprandial
about 10-15%.
If the concentration is lower than the reference interval, the condition is recognized as hypoglycemia.
It is a pathological state, during which the organism is not able to keep glucose homeostasis. Clinical
symptoms of hypoglycemia are manifested, when the level of glucose drops in adults below
2.8 mmol/L. Hypoglycemia is dangerous mostly because of the insufficient energetic supply into the
brain with symptoms like feeling hungry, headache, somnolence, mental perplexity, hallucinations and
finally spasms and coma. The second group of symptoms results from the activation of the adrenergic
system and the increase of the catecholamine secretion (palpitations, anxiety, tremble, perspiration).
Clinically, we distinguish between hypoglycemia developing after starvation and postprandial
hypoglycemia (develops after the meal). Hypoglycemia is evoked by a number of pathological
conditions, e.g. pancreatic tumours, defects in the production of anti-insulinic hormones, hepatic
cirrhosis, defects of enzymes metabolising glucose. Moreover, hypoglycemia develops after the
inadequate dose of insulin or peroral antidiabetics.
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Levels greater than reference are described as hyperglycemia. The most frequent and the most serious
cause of hyperglycemia is DM.
The level of fasting plasma glucose (FPG) is a good parameter for
diagnosis of diabetes mellitus
seeking persons with high risk of diabetes mellitus
Glycemia for diagnosis of diabetes mellitus (DM)
Diabetes is confirmed if glycemia exceeds:
combination of clinical symptoms with random determination of plasma glucose ≥ 11,1 mmol/l
the concentration of fasting plasma glucose ≥ 7 mmol/l
the concentration of glucose in glucose tolerance test ≥ 11,1 mmol/l
To confirm the diagnosis of diabetes, it is necessary to check the result repeatedly during following
days.
Seeking persons with high risk of diabetes mellitus
The high risk of diabetes is characterised by the value of fasting plasma glucose in the range 5,6-7,0
mmol/l. This state (IFP-impared fasting glucose) is known also as prediabetes.
Critical levels of fasting plasma glucose (FPG):
FPG (mmol/l) Interpretation
≤ 5,6 excluding DM
5,6 – 7,0 prediabetes (increased FPG, IFG)
≥ 7,0 DM
1.2.2 Determination of glycemia by personal glucometer
Quick and easy monitoring of glycemic profiles by patients themselves (self-monitoring) or directly at
the patients’ bed (bedside diagnostics) is carried out with the help of a large number of different types
of glucometers. Nowadays, there are given preferences to glucometers with broadened functions such
as: indication of measured values pre- and postprandial, setting sound signal for the remainder of next
determination of glucose, saving of dates to the memory of glucometer, because it is convenient for
monitoring of glycemia in diabetes. To compare measured values in capillary blood directly with
laboratory values it is suitable to use glucometer calibrated directly to the plasma. Capillary blood is
collected from the finger, sometimes alternatively collection is recommended (forearm, arm, calf etc.).
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Glucometers are mostly based on reflectance photometry: the sequence of reactions with glucose
oxidase and peroxidase proceeds on the indication zone of the diagnostic strip specific to the given
type of glucometer. The concentration of formed colour product is determined by reflectance
photometry. The new types of glucometers are based on electrochemical method when the weak
electric current formed during oxidation of glucose is detected.
Materials: Personal glucometer with diagnostic strips, blood collection strips, lancets
Procedure
Strictly follow the rules for the proper collection of capillary blood (see Exp. 1.1.1.2) and the
detailed instructions to glucometer.
Capillary blood collection: • wash hands in warm water
• disinfect the puncture site
• point the puncture using of lancet from lateral side of finger
• wipe off the first drop of blood
• support formation of the other drops by gently pressure to the surrounding tissue
• after blood collection apply a cotton wool wetted in disinfect solution on the
puncture site
Apply the drop of blood directly on the paper of test strip.
Insert the test strip into glucometer and after the required time read the value on the display.
Evaluation
Personal glucometers provide the patient the possibility to monitor and note down the glycemic profile
during the whole day, which is important for the proper treatment. Levels, which do not exceed
7 mmol/L, are regarded as satisfactory.
The determination of glycemia by glucometer can be used for diabetes/prediabetes screening program.
Generally, we may screen for elevated blood glucose with either a Fasting Plasma Glucose
(venipuncture) or Fasting Capillary Blood Glucose (home/fingerstick) test. It is necessary to confirm
the elevated blood glucose (determined randomly during day, independently on food intake) in
capillary blood above 7 mmol/l in physician´s office and then to confirm diagnose based on analysis of
clinical symptoms together with the examination of glycemia in venous blood by standard method in
laboratory.
1.2.3 Oral glucose tolerance test (oGTT)
If we find increased concentration of fasting glucose in the blood plasma, within 5,6–7 mmol/l, that is
known as a prediabetes, we should verify the effectiveness of glucose metabolism by the functional
test. However, oral glucose tolerance test is also performed when fasting plasma glucose is less than
5,6 mmol/l that is in this cases regarding persons with higher risk of diabetes or in the case of disorder
of tolerance glucose from the previous measurements.
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The glucose tolerance test monitors changes in glycemia after the peroral administration of the
standard dose of glucose by mouth. The oGTT test is not necessary at higher glucose concentrations,
especially if the characteristic symptoms of diabetes are present.
Standard procedure of oGTT in adults. Three days before the test saccharides are not restricted in the
diet. After a night fasting (10–14 hours), a sample of venous blood is taken. Then the patient is asked
to drink 75 g of glucose in approximately 300 mL of tea and venous blood sample is taken 2 hours
after drinking the tea. Patients are not allowed to smoke, eat or drink or do any physical activity.
Glyceamia is determined in plasma of venous blood
Materials: 75 g of glucose dissolved in 300 ml of weak tea. Personal glucometer with diagnostic strip, blood collection kit.
Procedure
Due to time-consuming and laboratory demands we will carry out oGTT only theoretically.
Despite this fact we will perform at least approximately this test for understanding of regulatory
mechanism of glucose and we will determine glycemia by glucometer.
Determine the concentration of glucose in capillary blood by personal glucometer (see Exp.1.2.2.)
in healthy volunteer at the beginning of the lesson (ideally at fasting state).
After drinking about 300 mL of tea with 75 g of glucose, repeat the determination of glucose
concentration in capillary blood by personal glucometer after 2 hours. For better understanding of
the regulatory mechanisms at healthy subjects there is recommended the determination of the
glucose concentration in capillary blood 30 minutes after drinking the tea.
Evaluation
Glycemia in plasma venous blood after 2 hours post-glucose (75 g) load (mmol/l)
Glucose tolerance Glycemia 2 hours after glucose load
Normal (exluding DM) < 7,8
Impaired glucose tolerance 7,8 - 11
Diabetes mellitus > 11
To confirm diagnose the result of test has to be check repeatedly. When finding impaired glucose
tolerance, o-GTTshould be repeated at two-year intervals.
► Evaluate the theoretical oGTT values given by teacher.
► Evaluate values obtained in the practical lesson by glucometer and note the trend of the
changes of glycemia during the time.
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1.2.4 Test for glucose in urine
Under normal conditions, there is only very small amount of glucose and other saccharides (galactose,
fructose, pentoses, lactose, and maltose) in urine, depending on the composition of the food eaten.
This concentration is undetectable by routine tests Glucose concentrations in urine higher than
0.8 mmol/L are considered as glucosuria. When the maximal reabsorption capacity of the cells of the
proximal tubule for glucose is exceeded, the resulting hyperglycaemia is manifested as glucosuria.
This value is usually given as 10 mmol/L (so-called renal glucose threshold), however, it may vary in
relatively large range (2.8–18 mmol/L). Rarely renal glucosuria appears during normal glycemia and
it is caused by disrupted tubular reabsorption.
Glucosuria usually reveals diabetes; however, if glucose is not present in urine, diabetes is not
excluded. Quantitative determination of glucosuria is not so appreciated in recent days it is used as
complementary test. It is important for evaluation of daily losses of glucose in urine.
Determination of glucosuria can be achieved by a non-specific test with Benedict’s reagent (see
Biochemistry II– practicals, Lesson 3, Exp.3.3.1) These tests are used only exceptionally. Specific
proof for glucose in urine can be carried out by diagnostic strips, e.g. glukoPHAN.
Quantitative determination of glucose in urine is performed using the same methods as for the
determination of glucose in plasma.
1.2.4.1 Test for glucose by diagnostic strip GlukoPHAN
Principle: The principle is the same as that in the enzymatic determination of glucose (see Exp. 1.2.1).
The indicator zone of the diagnostic strip contains the enzymes glucose oxidase and peroxidase and a
chromogenous substrate, which is oxidized in the presence of glucose by forming peroxide to a
coloured product.
Materials: Diagnostic strips GlukoPHAN.
Procedure
Dip the test strips in urine samples for about 1 s and wipe off the excess liquid on the vessel edge.
Put the strips onto the containers with urine (horizontal position of the strip).
After 3 minutes, compare the colour of the indicator area with the colour scale on the container.
Verify the possibility of false positive result by repeating the test with a new strip and urine sample
(with no proved glucose), which is measured into a test tube rinsed with a small amount of 3%
hydrogen peroxide or 0.5% Persteril
Evaluation
The test is quite sensitive. The result is clearly positive by the glucose concentration of 2 mmol/L. It is
very specific as well; other saccharides than D-glucose do not react.
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False negative results are created during high concentrations of reducing substances (ascorbic acid or
some spasmolytics slow down the development of the coloration).
False positive results can be created by peroxidase substrates present in the containers for urine
(common disinfectant Persteril, i.e. peroxoacetic acid, or hydrogen peroxide). Therefore, the
containers must be carefully washed by pure water after disinfection.
The use of the reduction test with Benedict’s reagent: If any of the hereditary disorders of saccharide metabolism (e.g.
galactosemia) are suspected, newborn departments request determination of other sugars than glucose. Comparison of
glycosuria examined by the enzymatic and reduction tests is carried out. If any difference is found, specific examination of
the presence of other monosaccharides must be done.
1.2.5 Test for ketone bodies in urine
Ketone bodies should be tested in urine of mainly diabetics of the 1st
type. If a diabetic undergoes
correct treatment, ketone bodies are not found in urine. Their presence, together with marked
hyperglycemia and glucosuria, indicate diabetic ketoacidose.
Principle: Ketone bodies react with sodium nitroprusside in alkaline medium to form a violet
coloured product. Test strips are based on this principle. We can use any of the test strips with a test
zone for detection of ketone bodies, e.g. diaPHAN. This test proves especially acetoacetate in fresh
urine, which during longer standing spontaneously decarboxylates to acetone (the test is less sensitive
for acetone).
Materials: Diagnostic strips diaPHAN.
Procedure
Dip the strip in urine sample for about 1 s and wipe off the excess liquid on the vessel edge.
After 1 minute, compare the colour of the indicator area with the colour scale on the container.
Evaluation
The excretion of ketone bodies in urine (ketonuria) is negligible under normal conditions; it does not
reach 0.5 mmol/day. A sharp increase in ketone bodies formation is a result of the increased utilization
of lipids (without providing enough glucose to be metabolized), as may occur in diabetes mellitus,
exhaustive physical activity without sugar delivery, prolonged starvation, reduction diet with protein
domination. In such cases, the concentration of ketone bodies in the blood rises above 200 µmol/L and
more than 100 mmol of ketone bodies may be excreted in urine daily. Acetone is relatively volatile
and it is expelled relatively quickly from the body through exhalation. Ketonuria with concurrent
glucosuria reflects metabolic decompensation of diabetics. Therefore, it is important to test ketonuria,
especially in every case of detected glucosuria.
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