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Theoretical part
Red blood cell count
Blood and its components
Blood, one of the bodily fluids, consists of blood elements (so-called formed elements or
corpuscles) suspended in blood plasma. Blood is highly specialised tissue, representing
approximately 7–8% of body mass. The volume of blood circulating within the cardiovascular
system in an average adult is roughly 4–6 litres, depending on gender, weight, body
consistency, etc.
Blood carries out a wide range of essential functions. It represents the main transporting
system supplying oxygen and nutrients throughout the body. Furthermore, blood removes
metabolic waste products such as lactic acid, carbon dioxide and so forth. Blood also has an
essential role in maintaining homeostasis, thermoregulation, immune reactions as well as
hormonal signalling and regulation.
Blood plasma is composed of water and organic (proteins, lipids, carbohydrates, etc.) and
inorganic substances (electrolytes) dissolved in the water. Blood plasma is obtained by
centrifugation of anticoagulated blood with consequent separation of its components,
separated based on their molecular weight. Anticoagulated blood is obtained by addition of an
anticoagulant agent into the blood which must be used in the production of blood plasma.
Inversely, blood serum lacking clotting factors is produced by centrifugation of blood without
any anticoagulation agent (clotted blood). The anticoagulant agents prevent a coagulation
cascade in any of its steps. Sodium citrate, EDTA and sodium oxalate bind calcium ions,
crucial reactants in the coagulation cascade and hence are used in vitro, in contrast to heparin,
which is used only in vivo. Heparin promotes the action of the anticoagulation systems,
especially antitrombin III.
Blood is ranked among the non-Newtonian fluids and thus its viscosity (the measure of a
fluid's ability to resist gradual deformation by shear or tensile stresses) is dependent on the
shear rate as well as its content, which highly influences its flow. The haematocrit represents
the volume percentage of red blood cells in blood and is examined by centrifugation or
sedimentation. The physiological range is normally 39–51% in men and 35–46% in women.
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Leukocytes
Leukocytes, also called white blood cells, are blood elements with a nucleus providing the
body’s immune response. The white cell count ranges under physiological conditions between
4000 and 10,000 leukocytes per 1 µl of blood, far less than the number of erythrocytes.
Commonly white blood cells are divided into two major groups, granulocytes and
agranulocytes. Granulocytes are further subdivided into neutrophils, eosinophils and
basophils, all representing the cellular subsystem of non-specific immunity. The
agranulocytes group comprises lymphocytes and macrophages.
Thrombocytes
Platelets (thrombocytes) are nuclei lacking discoid blood elements. They circulate in the
blood in the range of 150,000 to 450,000 per 1 µl of blood. Thrombocytes are essential in
haemostasis, a sophisticated mechanism preventing blood loss during injury. Moreover,
platelets play a crucial role also in coagulation. Platelets are produced in the bone narrow as
fragments of the megacaryocyte’s cytoplasm into circulation and after 7–10 days are
eliminated.
Erythrocytes
Erythrocytes, also called red blood cells, are blood elements produced in the bone marrow. As
they mature, erythrocytes lose their nucleus and take on a specific shape: oval biconcave
discs. Under the microscope one observes a brighter middle disc surrounded by a reddish
oval. This colour discrepancy is based on differences in haemoglobin concentration within the
erythrocyte. The bright central part of the erythrocyte is thinner (0.8 µm) and contains far less
haemoglobin in comparison to the periphery (2.2 µm). A red blood cell (RBC) with a 7.2 µm
diameter is called a normocyte. Larger RBCs are called macrocytes and smaller ones are
termed microcytes. The condition when there are a lot of RBCs of differing diameter is
known as anisocytosis. Poikilocytosis, on the other hand, stands for a condition where there
is an excessive amount of improperly shaped RBCs. 1 µl of blood contains 4.3–5.3 * 106
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RBCs in healthy men and 3.8–4.8 * 106
RBCs in women. A decreased number of RBCs is
called anaemia while an increased number is known as erythrocytosis or polyglobulia.
Erythropoiesis
Erythropoiesis is the specific process taking place within the bone marrow of adults which
leads to production of erythrocytes. It starts with a pluripotent stem cell that, under the effect
of erythropoietin, the red blood cell growth factor produced mainly in kidneys, differentiates
into a reticulocyte. The reticulocyte is eventually washed out into the blood, where 48 hours
later it develops into a mature erythrocyte. Physiologically the level of reticulocytes in the
blood is 0.5–1.5%, but this might be increased after an injury or other blood loss
(reticulocytosis). The normal life span of an erythrocyte is approximately 120 days. An old or
damaged erythrocyte is eliminated in the spleen.
Erythropoiesis requires several absolutely essential substances, such as:
1. Amino acids – an essential part of the globin part of haemoglobin as well as of
porphyrin
2. Iron – invaluable for hem production; a lack of it leads to iron deficiency anaemia
3. Vitamin B12 and folic acid – both crucial for nuclide acid synthesis and a lack of
them causes pernicious anaemia
Haemoglobin
Haemoglobin is a protein compounded from four heme molecules (a porphyrin ring
containing Fe2+
ion in the centre) and four globin molecules (two alpha and two beta chains).
Haemoglobin not only represents the most essential transport mechanism for oxygen, but it
also participates in CO2 transport. Each globin molecule creates a hydrophobic pocket
protecting a Fe2+
ion against its oxidation into a Fe3+
ion that would be incapable of further
transporting oxygen. Haemoglobin is produced during the evolution of an erythrocyte from its
precursors in the bone marrow from glycine and succinyl-CoA.
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Haemoglobin forms Bound molecule
Oxyhaemoglobin O2
Carbaminohaemoglobin CO2
Methaemoglobin Heme contains Fe3+
instead of Fe2+
Carboxyhaemoglobin CO
The initial phase of haemoglobin degradation is located within macrophages following
phagocytosis of a disrupted erythrocyte or the haemoglobin molecule leaking into plasma and
bound to haptoglobin, a protein binding free haemoglobin molecules floating in plasma.
Degradation of haemoglobin is caused by its oxidation into verdoglobin. Verdoglobin is
transformed to green biliverdin by splitting the globin molecule off, which is eventually
chopped off into amine acids. Biliverdin is enzymatically modified and, as bilirubin, it is
released into the blood stream. Bilirubin as a lipophilic molecule is bound to albumin,
transporting it to the liver where it is further modified by conjugation with glucuronic acid.
Conjugated bilirubin, so-called direct, is excreted into the gall and consequently mixed with
chymus. An increased bilirubin concentration in plasma is known as jaundice or icterus and
typically first appears as yellowish discolouration of sclera and eventually mucosa and skin
accompanied by itching.
Sex differences
Men Women
total red blood cells 4.3–5.3 x 1012
/l 3.8–4.8 x 1012
/l
hemoglobin 140–180 g/l 120–160 g/l
hematocrit 0.39–0.51 0.35–0.46
Sex differences are caused by sex hormones. Testosterone stimulates erythropoiesis by
increasing erythropoietin production in kidneys, while oestrogen decreases its production.
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Anaemia
Anaemia is a medical condition where haemoglobin concentration is decreased, causing
insufficient oxygen transport throughout the body. Anaemia develops when the production of
red blood cells is impaired or loss of them increases. Anaemia might present as pale and cold
skin, shortness of breath, tiredness and poor physical endurance. The body compensates for
anaemia by increasing the heart rate.
Iron deficiency anaemia
Iron deficiency anaemia is the most common anaemia in humans, predominantly occurring in
women and caused by a lack of iron necessary for haemoglobin production. Hypoxemia
caused by anaemia stimulates massive erythropoietin production and thus the erythrocytes
released from the bone marrow are small and have a decreased haemoglobin content
(microcytic hypochromic anaemia).
Pernicious anaemia
Pernicious anaemia is a medical condition that develops due to a lack of vitamin B12 or folic
acid. Both of these vitamins are crucial for the nuclide acid production, namely thymine.
Because there is not enough thymine for DNA synthesis, erythrocytes produced in the bone
marrow are great and contain a lot of haemoglobin (macrocytic, hyperchromic anaemia).
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Protocol
Red blood cell count
Methods
Red blood cell count
Equipment: Bürker’s counting chamber with cover glass, microscope with lamp, a cup of
approx. 10–12 ml with stopper, micropipette with adjustable range of 1–5 ml and 10–100 μl,
dropper with a fine tip, blood sample, Hayem’s solution – beware! POISON! (Na2SO3 – 5 g,
NaCl – 1 g, HgCl2 – 0.5 g, distilled water 200 ml), a vessel with disinfecting solution.
Note:
1. Hayem’s solution is a highly toxic solution due to its HgCl2 content, which causes
burns after ingestion and is harmful also after long-lasting skin exposure. Before
handling, read the safety rules carefully!
2. Always use gloves when working with blood!
Procedure:
1. Place 4950 μl of Hayem’s solution into the cup by using the micropipette.
2. Add 25 μl of blood using the other micropipette: the chosen volume (25 μl) is set on the
micropipette and can be checked on its display. Fix the tip by slightly pressing the bottom
of the micropipette, immerse it (approx. 1 cm) into the blood sample and gently press the
button (position 1). Pull the tip from the blood – any excess of blood should be carefully
removed using a pad of cotton wool. Then, the blood is expelled into the cup with
Hayem’s solution. The button of the micropipette is pressed completely (position 2) when
the tip is immersed in the solution – the whole volume of blood is expelled. Blood is
slowly expelled into the solution, and near the wall of the vessel so that it is not mixed
yet. The used tip is then disposed into the vessel with disinfecting solution by pressing
the ejector of the micropipette, and the device is then placed back onto the stand.
3. The cup is closed by a stopper and its content mixed by whirling movements for 1–2 min
in order to get a homogenous suspension. The solution should not come into contact with
the stopper.
4. Prepare the Bürker’s counting chamber. On the middle prism, two grids are engraved
constituting a network of smaller (1/400 mm2
) and larger (1/25 mm2
) squares for
counting red and white blood cells, respectively.
5. Take a sample of the suspension from the cup into a fine dropper. The tip of the dropper
is placed between the cover glass and the bottom of the chamber where the suspension is
drawn by capillarity. When filling the chamber, we have to prevent the solution from
overflowing into the grooves between the prisms or to the space over the other grid where
the red blood cell count should be performed. If this happens, the chamber must be
cleaned and refilled. Then, put the filled chamber on the microscope plate and, under
steady visual control, bring the objective near to the cover glass.
6. Red blood cells are counted in a total of 40 (or 80 if a higher precision is desired) small
squares. If some cells touch the margin of the square, only those lying on the upper and
the right side of the square are counted, irrespective of whether they are within or outside
the square, as shown in Fig. 2.
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7. Estimate the average number of RBCs in one square from the value obtained by counting
40 or 80 squares. Since the volume above the square is 1/4000 mm3
, multiply the average
number by 4.109. Finally, multiply by the dilution ratio, i.e. 199 to get the number of
RBCs in 1 litre of blood. The margin of error of such estimation of RBCs is
± 200,000 mm3
.
The Bürker’s counting chamber and basic rules for counting RBCs
1/400 mm2
Fig. 1 Fig. 2
Estimation of haemoglobin concentration
Equipment: Spectrophotometer Spekol, funnel and 5 ml cylinder for transforming solution,
micropipette with adjustable range 10–100 μl, vessel with disinfection solution, and
transforming solution (solution by van Kempen-Zijlstra: K3Fe(CN)6 – 0.2 g, KCN – 0.05 g,
KH2PO4 – 0.14 g, distilled water to 1000 ml; in Drabkin’s solution, KH2PO4 is replaced by
NaHCO3 – 1.0 g), automatic analyser Mythic 18
Note:
1. Transforming solution is a highly toxic solution due its KCN content, which is highly
toxic when inhaled, ingested or in contact with skin. Before handling, read the safety
rules carefully!
2. Always use gloves when working with blood!
Procedure:
1. Switch the apparatus on and wait at least 15 minutes before the measurement. For
estimation of haemoglobin concentration, a wavelength of 540 nm is used. The lower
lever has to be turned to the left position.
2. Prepare the measured solution: put 5 ml of transforming solution into a test tube by using
the funnel and 5 ml cylinder and add 20 μl of blood taken from the blood sample by
means of an adjustable micropipette (for the procedure, see Exercise I.1). The absorbance
is measured 10 min after mixing the solution.
3. Fill the cuvette with distilled water (blind experiment) and move cuvette inside the
apparatus. Press C. Press FAKT – either 1.000 or last factor will appear on the display.
Insert the correct factor for your measurement (for haemoglobin this is 22.8) using POS
and INC. Press FAKT again (this inserts the value into the memory of the Spekol). Press
R. Now you can read the concentration of the blind experiment.
1/25 mm2
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4. After this setting, insert the cuvette with the measured sample and read the concentration
of haemoglobin (mmol/l). Multiply this value by the coefficient 16.11 to obtain the
concentration of Hb in g/l.
5. Check the values obtained by the Spekol by using the Mythic 18 automatic analyser.
Note:
Cuvettes must be clean and their outer surface dry. If some solution gets onto the surface
while filling the cuvette, it must be wiped off (the instrument would be damaged if the
aggressive solution reaches its inside). Cuvettes may be taken only by their upper parts,
optimally in lateral ground areas, never in areas where the measuring light beam passes. The
meter and the fragile cuvettes are the most sensitive parts of the instrument: avoid any shock
or shaking.
Calculated parameters of red blood cells
Based on the values obtained by measurements, further parameters commonly used in clinical
practice can be reached by calculation:
1. average volume of red blood cell (MCV = mean corpuscular volume)
MCV = Hct / number of red blood cells (physiological values: 80–95 fl)
2. average weight of Hb in red blood cell (MCH = mean corpuscular haemoglobin)
MCH = Hb / number of red blood cells (physiological values: 27–32 pg)
3. average concentration of haemoglobin in red blood cell (MCHC = mean corpuscular
haemoglobin concentration)
MCHC = Hb / Hct (physiological values: 310–360 g Hb / litre of red blood cell)
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Results
Red blood cell count
Describe the method:
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Fill in the number of red blood cells (counted in 40 small squares)
.... ..... ..... ..... ..... ..... ..... ..... ..... .....
.... ..... ..... ..... ..... ..... ..... ..... ..... .....
.... ..... ..... ..... ..... ..... ..... ..... ..... .....
.... ..... ..... ..... ..... ..... ..... ..... ..... .....
Average number of red blood cells per square
.............................................................
The volume of one small square: 1/4000 l
The number of red blood cells in one small square is multiplied by 4 * 109
............................................................
Obtained value is multiplied by * 200
Total red blood cell /l:..........................................
Estimation of haemoglobin concentration
In Graph 1 note the haemoglobin concentration that you have found. The physiological range
of haemoglobin concentration in men 130–175 g/l [Hb (1Fe) 8.07 – 10.9 mmol/l], and in
women 120–165 g/l [Hb (1Fe) 7,45 – 10,2 mmol/l], is marked in the graph.
Declined values: anaemia; raised values: dehydration, polycythaemia
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Graph 1. Haemoglobin concentration (g/l)
g/l man woman
180
170
160
150
140
130
120
110
100
90
Calculated parameters of red blood cells
1) Average volume of red blood cell (MCV = mean corpuscular volume)
MCV = Haematocrit / number of red blood cells (physiological values: 80–95 fl)
Calculation: MCV =
2) Average weight of Hb in red blood cell (MCH = mean corpuscular haemoglobin)
MCH = haemoglobin / number of red blood cells (physiological values: 27–33 pg)
Calculation: MCH =
3) Average concentration of haemoglobin in red blood cell (MCHC = mean
corpuscular haemoglobin concentration)
MCHC = haemoglobin / haematocrit (physiological values: 310–360 g/l)
Calculation: MCHC =
Conclusion
According to your results, draw a conclusion about your specimen. Suggest the gender of
your unknown patient and attempt to diagnose him/her.
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