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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.
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
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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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Theoretical part
Determination of blood groups by slide
method
Introduction
The erythrocyte cytoplasmic membrane serves as a barrier between the interior and blood
under physiological conditions. Furthermore, it has a profound impact on intercellular
communication and interaction. The cytoplasmic membrane is typically organised into a
phospholipid bilayer with hydrophobic regions closed in on each other and hydrophilic
regions interacting with the inner and outer environment. Within the phospholipid bilayer
there are embedded proteins as well as lipids (mainly cholesterol) which reinforce the whole
structure. A sort of plasma proteins, carbohydrates, glycolipids and glycoproteins stretches
out beyond the membrane into the outer environment as antigens, based on which the wide
range of blood groups is determined, such as AB0, Th, MNs, Kell, Levis, and so on. A blood
group is inherited co-dominantly and remains unchanged over one’s entire lifetime.
AB0 system
The AB0 system represents the most examined blood group, and is determined by the
presence of the antigens A and B. These antigens are chemically glycoproteins and differ in
their terminal carbohydrate chains, so-called agglutinogens. Four blood types are
distinguished:
A – only agglutinogen A is present on the erythrocyte’s surface
B – only agglutinogen B is present on the erythrocyte’s surface
AB – agglutinogens A and B are present on the erythrocyte’s surface
0 – neither agglutinogen A nor B is present on the erythrocyte’s surface
Blood circulating throughout the body contains immunoglobulins IgM (anti-A and anti-B)
known as agglutinins. Production of these antibodies is triggered by one’s first intake of food
and lasts throughout one’s entire lifetime.
Group A – produces only anti-B agglutinin
Group B – produces only anti-A agglutinin
Group AB – no agglutinins are produced
Group 0 – produces both anti-A as well as anti-B agglutinin
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The interaction of agglutinogen and agglutinin leads to agglutination, production of
floating flakes accompanied by subsequent destruction of erythrocytes by the complement
system.
Explanation of slide method
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As an example to explain the slide method, we will look at the group A. In the blue drop
containing anti-A agglutinins, there is an obvious reaction of anti-A agglutinins and red blood
cells holding A agglutinogens on their surface lead to the production of large erythrocyteimmunoglobulin
complexes. This reaction cannot be observed in the yellow drop owing to the
lack of agglutinin anti-B which does not have its agglutinogen B counterpart on the
erythrocyte’s surface. In the last grey drop agglutination is displayed due to the presence of
both anti-A and anti-B agglutinins, but only anti-A agglutinins take part in this reaction.
Hereditability of the AB0 system
Blood groups are inherited from both parents. The AB0 blood type is determined by a single
gene producing three alleles – allele A, B and i. A specific dominance relationship occurs
called codominance where allele i is recessive to the dominant A and B alleles.
Phenotype – blood group Alleles
A AA, Ai
B BB, Bi
AB AB
0 Ii
Rh factor
The Rh factor represents another blood group determined by antigens C, c, D, d, E, e.
Nevertheless, the Rh factor is defined by the presence or absence of the antigen D. A Rh
positive subject is a carrier of antigen D on the surface of red blood cells and vice versa.
Antigen D is dominantly inherited and thus a Rh negative subject has inherited the recessive
allele from the both parents. Contrary to the AB0 system, antibodies against antigen D are
produced just after the Rh negative subject’s first encounter with antigen D – this reaction is
called immunisation. Moreover, anti-Rh antibodies are produced as IgG type, and thus are
capable of being transported through the placental barrier.
Haemolytic disease of newborns (Erythroblastosis fetalis)
This disorder occurs in Rh negative mothers immunised against the Rh factor (i.e. there are
anti-Rh factor antibodies circulating in her blood) and carrying a Rh positive baby. The
mother’s immunisation might be result of either a previous pregnancy when the mother’s and
a Rh positive baby’s blood were mixed or by administration of an incompatible blood type.
The subsequently created anti-D antibodies can be transported through the placental barrier
and lead to destruction of red blood cells in the newborn. An elegant method of preventing the
production of anti-D antibodies has been introduced: after each delivery of a Rh positive
baby, anti-D immunoglobulin is administered to the Rh negative mother in order to destroy all
Rh positive erythrocytes leaked during the labour.
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Theoretical part
Erythrocyte sedimentation rate
Blood plasma
Blood plasma is an amber solution containing dissolved organic as well as inorganic
compounds. Plasma represents 5% of the body mass, which stands for 3.5l in an average man
weighing 70 kg. Blood plasma is obtained by the centrifugation of anticoagulated blood, thus
it contains clotting proteins in comparison to the blood serum.
The organic compound of blood plasma is made up from proteins, glucose, hormones,
vitamins, enzymes etc. The plasmatic proteins can be further divided into an albumin,
globulin and fibrinogen fraction. The physiological concentration range of the plasma proteins
is 65–85 g/l, but the vast majority is represented by albumin (25–51 g/l). The capillary wall is,
under physiological conditions, a completely impenetrable barrier for the proteins dissolved in
plasma and hence an osmotic pressure of around 25 mmHg is created. Furthermore, the
proteins represent a valuable buffer system maintaining the pH of blood plasma in the very
narrow range 7.36-7.44, a fast source of amino acids, they play an important role in immune
reactions, coagulation, transportation of substances such as fatty acids, steroid hormones,
bilirubin, ions, metals, drugs and so forth.
The inorganic compound of blood plasma consists of dissolved ions.
Ion Concentration
Sodium 135–145 mmol/l
Potassium 3.7–5.1 mmol/l
Chloride 96–108 mmol/l
Calcium 2–2.6 mmol/l
Calcium ionized 1.16–1.32 mmol/l
Inorganic phosphates 0.65–1.61 mmol/l
Magnesium 0.78–1.03 mmol/l
Erythrocyte sedimentation rate (FW)
The erythrocyte sedimentation rate (Fahraeus-Westergren) is a nonspecific laboratory
method providing information for the caregiver that something is going on in the patient’s
body. Sedimentation is a process of unsolvable particles settling in the suspension. Its
counterpart is suspension stability, which is represented by zeta potential, the negative charge
on the surface of erythrocytes determined by sialic acids bound on glycophorin which repel
erythrocytes away from each other. This procedure is traditionally performed as
anticoagulated blood placed in an upright tube and the erythrocyte sedimentation rate is
measured after one hour.
Factors influencing the erythrocyte sedimentation rate:
• Red blood cell diameter – the bigger the red blood cells are, the faster sedimentation is
• The number of erythrocytes – more erythrocytes means greater zeta potential and
higher suspension stability
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• The plasmatic proteins
 Albumin – possess negative charge, and this leads to higher suspension
stability
 Fibrinogen – is negatively as well as positively charged and so suspension
stability is decreased
 Immunoglobulins – also hold negative and positive charge and hence lower
suspension stability
Factors disrupting the zeta potential lead to rouleaux production. Roleaux sediments faster
because of increased ratio of volume/surface area.
Erythrocyte sedimentation rate is measured in anticoagulating blood produced by addition of:
 Natrium citrate – binding Ca2+
 EDTA - Ethylenediaminetetraacetic acid – this also acts as a chelating agent
 Natrium oxalate – the same as EDTA
 Heparin – activation of antitrombin III
Physiologic values:
 Man – 2–8 mm/h.
 Woman – 7–12 mm/h.
 Newborn – 2 mm/h
 Toddler – 4–8 mm/h.
→ the gender differences are caused by sex hormones; testosterone increases the erythropoietin
production in kidneys → more erythrocytes
Pathologies:
 Increased erythrocyte sedimentation rate:
 Pregnancy
 Macrocytosis
 Infection
 Tumours – myeloma
 Inflammations
 Necrosis (infarction, trauma)
 Relative/absolute albumin loss (nephrotic syndrome)
 Decreased erythrocyte sedimentation rate
 Misshaped erythrocytes – spherocytosis
 Polycythemia vera
 Leucocytosis
 Dysproteinaemia – hypofibrinogenaemia, hypogammaglobinaemia
 Dehydration
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Theoretical part
Osmotic fragility test
The cytoplasmic membrane of red blood cells
The red blood cell cytoplasmic membrane not only represents a barrier between the interior of
the erythrocyte and the outer environment, it also plays a crucial role in intercellular
communication and adhesion. Some of its important features are deformability, flexibility as
well as durability which allows cells to move through capillaries (the diameter of which can
be as narrow as half of the diameter of the erythrocyte). Erythrocytes possess all these
features thanks to the cytoskeletal proteins where spectrin plays the central role. Spectrin fibre
is formed by spectrin alpha and beta and further interacts with actin filaments to create an
intracytoplasmic cytoskeleton connected to the inner layer of the cytoplasmic membrane by
protein 4.1 and ankyrin. The older the erythrocyte gets, the less durability, flexibility and
deformability it possesses, and after approximately 120 days it is destroyed in the spleen.
Osmotic resistance
The resistance of erythrocytes to mechanical, chemical and osmotic damage is not identical in
all red blood cells, and therefore some are able to resist higher stress levels than others. This is
dependent on the cell’s age, shape (e.g. spherical), enzyme content (e.g. genetic deficiency of
glucose-6-P dehydrogenase), etc. The osmotic fragility test is a specific method used in the
differential diagnosis of haemolytic anaemias.
Diffusion Movement of particles based on the
concentration gradient
Osmose Movement of solvent molecules through the
semipermeable membrane into a region with
higher solute concentration
Osmotic pressure Pressure required to stop osmosis
Osmolarity Measured solute concentration defined as
the number of osmotic active molecules per
1 litre of its solvent
Osmolality Same as osmolarity but per 1kg; = 2
[Na+
]+[glc]+[urea] = 275-295mmol/kg H2O
Tonicity Represents the effect of osmotic activity in
the relationship with a cell
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Pathology:
 Elevated values of minimal osmotic resistance
 Inherent haemolytic anaemias
 Decreased values of maximal osmotic resistance
 Polycythemia vera
 Thalassemia
 Sickle-cell anaemia
 Fe2+
deficiency
 Status post splenectomy
Isotonic haemolysis
The isotonic haemolysis of red blood cells occurs under two specific conditions:
 Isotonic solution of glucose – glucose is absorbed by cells until the solution becomes
hypotonic and the cells die
 Isotonic solution of urea – urea freely penetrates cells following the concentration
gradient and at the same time the tonicity of the solution decreases (urea does not
respect the semi-permeability of the cytoplasmic membrane)