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I. Red blood cell count. Estimation of haemoglobin concentration. Calculated parameters of red
blood cells
II. Estimation of blood group by slide method
Physiology I – practice
Autumn, weeks 4-6
Fyziologický ústav LF MU, 2015 © Michal Hendrych

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Red Blood Cell Count
Department of Physiology, Faculty of Medicine, Masaryk university

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Red Blood Cell (RBC) – erytrocyte
̶Anucleated cell, the most abundant blood cell
̶Shape:
̶biconcave disc – surface increased by 30%
̶the shape is ensured by a protein named spectrin
̶the ability to deform inside the capillaries is necessary
̶Functions:
̶Transport of oxygen to tissues (bonded mainly to haemoglobin)
̶Role in acid-base balance and CO2 transport
̶Size:
̶Normocyte: 7.5 µm
̶Microcyte: ≤ 7 µm
̶Macrocyte: ≥ 9 µm
̶Megalocyte: ≥ 20 µm
̶The width is around 2.5 µm peripherally and 1 µm centrally

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Reticulocyte
̶Immature RBC
̶In peripheral blood: 1% ± 0.5% of RBC
̶reticulocytosis: increased ratio of reticulocytes in peripheral blood, occurring mainly after
blood loss or blood donation
̶No nucleus, but residues of membrane organelles in the cytoplasm (substantia granulo-filamentosa)
̶Within 48 hrs. maturation to RBC
̶
̶
retikulocyte
mature RBC

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Poikylocytosis: irregular shape of RBCs
̶Usually in anaemia

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Department of Physiology, Faculty of Medicine, Masaryk university
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RBC count
̶Number of RBC (red blood count)
̶Men: 4.3-5.3 * 1012 / l
̶Women: 3.8-4.8 * 1012 / l
̶Newborns: 4.4-7.0 * 1012 / l
̶Intersexual differences:
̶Men: testosterone (male sex hormone) stimulates releasing of erythropoietin
̶Women in fertile period: relative erythrocytopaenia due to menstruation (and lower levels of
testosterone)
̶Alterations of RBC count
̶Polyglobulia – increased number of RBCs, increased blood viscosity
polycythemia vera, dehydration, adaptation to high altitude
̶Erythrocytopaenia – decreased number of RBCs, decreased blood viscosity
̶

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Estimation of RBC count
̶Automatic methods
̶Electrical impedance – RBC count is determined based on the increase in electric resistance and
the decrease in electric current while passing through the capillary from the storage container to
the smaller container. RBC has a lower conductivity than diluent. It allows us to find out the size
of the RBC, small = higher current, large = lower current.
̶Photo-optical – when passing through the capillary, a light beam falls on the RBC and the RBC
causes the dispersion of light which we capture.
̶Classical method:
̶Bürker‘s chamber + Hayem‘s solution for dissolution
̶4950 µl of Hayem‘s solution and 25 µl of blood – dilution *198, or
̶4975 µl of Hayem‘s solution and 25 µl of blood – dilution *199
̶

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Haematocrit
̶The volume percentage of red blood cells in a blood sample (erythrocyte volume fraction)
̶Estimation after centrifugation of anti-coagulated blood*
̶Plasma
̶Buffy coat – white non-transparent layer above RBCs consisting of leukocytes and thrombocytes
making up 1% of the whole volume
̶RBC
̶Hct (hematocrit)
̶Men: 42-52%
̶Women: 37-47%
̶
̶•centrifugation of coagulated blood results in blood serum (which differs from plasma by the
absence of coagulation factors)

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Department of Physiology, Faculty of Medicine, Masaryk university
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Hematocrit
plasma (55%)
leukocytes and thrombocytes (1%)
erythrocytes (44%)
  Normal    Anaemic
blood       blood Polycythemia

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Hemoglobin
̶Structure:
̶Adult haemoglobin is comprised of α and β globin subunits
̶Fetal haemoglobin consists of α and γ globin subunits
higher affinity to oxygen than in adult hemoglobin
̶A heme group contains an iron ion (Fe2+) which is the site of oxygen binding
̶Hemoglobin concentration (HGB)
̶Men: 140-180 g/l
̶Women: 120-160 g/l
̶Newborns: 160-240 g/l
̶Spectrophotometric determination of HGB
̶An addition of the transformation solution to the blood causes the lysis of erythrocytes and the
release of haemoglobin and its transforms into cyanohaemoglobin. Measurement of light absorption
follows.

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Hemoglobin – derivates
̶Oxyhemoglobin – hemoglobin + O2
̶Bright red color
̶Carboxyhemoglobin – hemoglobin + CO
̶cherry red color, CO saturation higher than 50% results in a loss of consciousness
̶Carbaminohemoglobin – hemoglobin + CO2
̶dark red color, CO2 binds to an amino group at the N end of the protein chain. The
CO2 concentration in inhaled air higher than 5% may lead to an impaired consciousness because of
the disrupted binding of O2 to hemoglobin
̶Methemoglobin = hemiglobin with oxidized Fe (Fe3+)
̶dark "chocolate" brown, it does not bind O2,  1-3% levels are normal, 70% levels are fatal
̶Glycated hemoglobin
̶a molecule of glucose binds to the protein chain – it reflects long-term blood sugar levels
(glycemia). Normal levels are up to 4 mmol/l, increased levels are in uncompensated diabetes
̶

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Oxy and deoxyhemoglobin
arterial oxygenated  blood
venous deoxygenated  blood
Absorption spectra of hemoglobin derivates
oxyhemoglobin
deoxyhemoglobin
(without oxygen)
methemoglobin
carbonylhemoglobin
Spectra of oxyhemoglobin and carboxyhemoglobin are similar at the wavelength that evaluates the
saturation of hemoglobin with oxygen → hypoxia caused by CO poisoning cannot be detected this way
unless the sensor is specialized for it

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Department of Physiology, Faculty of Medicine, Masaryk university
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Hemoglobin derivates – methemoglobin
Higher methemoglobin concentration leads to the darker coloration of the arterial blood
Color spectrum for evaluation of metHb concentration
Oxygenated blood is used for the evaluation
High metHb concentration in blood
Normal blood

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Department of Physiology, Faculty of Medicine, Masaryk university
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Hemoglobin derivates – methemoglobin
̶Formation of metHb – nitrite reacts with the blood to create metHb
̶A blood enzyme named methemoglobin-reductase reduces Fe+3 back to Fe2+
̶Furthermore, non-enzymatic reduction via vitamin C is possible
̶Infants have insufficiently developed reduction mechanisms
̶(+ fetal hemoglobin is easily oxidized)
̶→ it is necessary to reduce intake of nitrites in the infant diet (use of infant water)
̶Methemoglobinemia – congenital or acquired causes
̶Congenital – impaired synthesis of enzymes
̶Gained – increased intake of nitrates and nitrites
  (fertilizers), poisoning (nitrobenzene, aniline), drugs
  (benzocaine, also phenacetin, sulfonamides)
̶Therapy – administration of reducing agents such as
   methylene blue or ascorbic acid
MetHb values
Symptoms
0–2%
normal values
<10%
cyanosis
<35%
cyanosis a others (headache, dyspnoea)
>70%
fatal

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Calculated parameters of RBCs
̶The average volume of one RBC (MCV, mean corpuscular volume)
̶MCV = HCT/RBC (hematocrit/red blood count) = 80-95 fL
̶The average weight of Hb in one RBC (MCH, mean corpuscular hemoglobin)
̶MCH = HGB/RBC (hemoglobin/red blood count) = 28-32 pg
̶The average concentration of Hb in one RBC (MCHC, mean corpuscular hemoglobin concentration)
̶MCHC = HGB/HCT (hemoglobin/hematocrit) = 310-360 g/L
̶Red cell distribution width (RDW) = 11,5-14,5%
̶Variation of RBCs size
̶↑RDW – anisocytosis

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Anisocytosis: varying size of RBCs
Mild anisocytosis is physiological

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Calculated parameters of RBCs
HCT
RBC
HGB
MCH = HGB/RBC
MCHC = HGB/HCT
MCV = HCT/RBC
hematocrit
men: 42-52%
women: 37-47%
hemoglobin concentration
men: 140-180 g/L
women: 120-160 g/L
number of RBCs
men: 4.3-5.3 * 1012/L
women: 3.8-4.8 * 1012/L
RBC volume
80-90 fL
Hb in RBC
310-360 g/L
Hb concentration in RBC
28-32 pg/RBC
↑MCV → macrocytosis
↓MCV → microcytosis
↑MCH → hyperchromic RBC
↓MCH → hypochromic RBC
Department of Physiology, Faculty of Medicine, Masaryk university

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Department of Physiology, Faculty of Medicine, Masaryk university
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Anemia
̶= Decreased concentration of functional Hb in the blood
̶A small number of RBCs or low hematocrit or unfunctional Hb
̶Impaired RBC production (proliferation or differentiation), increased RBC destruction or blood
loss
̶RBCs are one of the fastest proliferating cells within the body thus early signs of malnutrition
may be seen in RBC count
̶Anemia has various causes (congenital or acquired)
̶Symptoms (anemic syndrome)
̶Tissue hypoxia: pale skin and mucose membranes
  (best seen in the conjunctiva), fatigue, weakness
̶Tachycardia
̶Dyspnoea in mild physical activity
̶

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Department of Physiology, Faculty of Medicine, Masaryk university
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Sideropenic anemia
̶Cause: deficiency of Fe2+ → decreased production of hemoglobin → hypoxia stimulates releasing of
erythropoietin → increased production of RBCs with insufficient Hb levels
̶Microcytic hypochromic anemia
̶↓MCV and ↓MCH, RBC count increased or unchanged
̶RBCs are small and pale with Hb only peripherally

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Megaloblastic anemia
̶Megaloblasts (oversized immature RBCs) in the bone marrow and a reduced number of reticulocytes in
the peripheral blood
̶Macrocytic hyperchromic anemia (↑MCV, ↑MCH, ↓RBC)
̶A small number of large erythrocytes with an increased hemoglobin content
̶It occurs due to a lack of vitamins B12 and folic acid which are important for the methylation of
uracil to thymine
̶↓ B12 and folic acid → ↓DNA formation, while RNA and protein synthesis is intact
̶B12 supply - years; folic acid – months → manifestations of hypovitaminosis occur with time delay
̶Pernicious anemia – the most common type of megaloblastic anemia
̶Autoimmune disease interfering with B12 absorption
̶Autoimmunely damaged parietal cells of the gastric mucosa do not form intrinsic factor, which is
necessary for the absorption of B12 (a disorder of factor formation can also
   be due to gastric surgery)

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Megaloblastic anemia
megaloblast
Megaloblastic anemia
Norrmal blood smear
RBCs are larger, hyperchromic, their centre is less pale than in normal RBCs
decreased RBC count (+presence of hypersegmented neutrophils)
RBC
neutrophil
Megaloblastic  anemia
Normal blood
Department of Physiology, Faculty of Medicine, Masaryk university

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Department of Physiology, Faculty of Medicine, Masaryk university
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Blood groups (types)

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Department of Physiology, Faculty of Medicine, Masaryk university
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Antigens and antibodies in blood groups
̶Blood groups are determined by antigens on the RBC membrane
̶These blood traits are inherited and remain unchanged throughout life
̶A light introduction to the general principles of immunization
̶An antigen is a "recognition mark" by which the immune system determines whether it is a body’s
cell or a foreign one
̶When the immune system first encounters a foreign erythrocyte in the blood, it begins to produce
antibodies against the foreign antigens on the erythrocyte (but not against those antigens that are
the same as on the body’s erythrocytes). It remembers the foreign antigen and creates antibodies
faster the next time it encounters it.
̶If there are antibodies against a certain blood type in the blood, the erythrocyte of this blood
type triggers an immune reaction → destruction of the foreign blood cell (hemolysis)
̶

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Department of Physiology, Faculty of Medicine, Masaryk university
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Antigens and antibodies in blood types
̶The most important blood groups are AB0 and Rh
̶Antibodies against AB0 antigens are in the blood from the first months of life
̶Antibodies against Rh factor and other blood types form only after immunization with incompatible
blood – unwanted immune response during repeated transfusions of not fully compatible blood
̶There are dozens of other systems that need to be considered from an immunological point of view
(MNS, Kell, Lewis, Kidd systems, ...)
̶

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AB0 system
̶Antigens on the surface of RBCs (agglutinogens): A, B
̶Antibodies in the blood (agglutinins): anti-A, anti-B (IgM)
̶They do not cross the placenta
̶Agglutination (clumping):
̶anti-A reacts with A, anti-B reacts with B
̶RBCs are connected by agglutinogens – clumps are formed
̶An immune response is triggered (complement activation) that destroys foreign erythrocytes via
hemolysis
̶
̶Immunization against A and B happens during the first months of life (these antigens are also in
the diet) – agglutinins are then in the blood for the rest of the life
̶When incompatible blood is administered - strong activation of the immune system occurs, leading
to hemolysis, anemia, hemoglobinuria (hemoglobin in the urine), kidney failure, ... death
̶
agglutinogen
agglutinin

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Department of Physiology, Faculty of Medicine, Masaryk university
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AB0 system
Blood groups
Group A
Group B
Group AB
Group 0
Prevalence in CZ
41%
18%
9%
32%
RBCs
Antigens on  RBCs
A
B
A a B
none
Antibodies in the blood
anti-B
anti-A
none
anti-A + anti-B

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Department of Physiology, Faculty of Medicine, Masaryk university
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Rh factor
̶Antigens D, d (also C,c, E, e, which are weaker) are only on RBCs
̶The strongest one is an antigen D – if present → Rh+ blood group
̶In recessive homozygotes (dd) → blood group Rh- (17% in Europe, <1% elsewhere)
̶in Rh- blood, antibodies (anti-D, IgG) develop only after immunization (Rh- blood must meet Rh+
blood)
̶The first reaction is weaker, the next encounter with Rh+ blood will trigger a stronger immune
response → hemolysis
̶

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Department of Physiology, Faculty of Medicine, Masaryk university
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Rh factor
̶Causes of immunization:
̶Transfusion of incompatible blood – Rh- recipient receives Rh+ blood
̶Childbirth (miscarriage, abortion, bleeding from the placenta, invasive procedures) where the
mother is Rh- and the fetus is Rh+
̶Immunization will manifest only during further pregnancies of this combination – IgG antibodies
pass through the placenta and attack fetal RBCs → blood hemolysis → anemia → increased
hematopoiesis → fetal erythroblastosis (more erythroblasts – immature RBCs), unconjugated
hyperbilirubinemia (icterus), anemia, hydrops (tissue swelling due to hypoxia), death
̶Prevention – administration of anti-D after delivery (or other events) will eliminate Rh- RBCs
before the mother's immune systém notices them
̶

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Department of Physiology, Faculty of Medicine, Masaryk university
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Estimation of blood groups by slide method
̶Drop anti-A, anti-B and a combination of anti-A and anti-B antibodies onto the glass slide
̶Or blood group sera A (contains anti-B), B (anti-A) and 0 (anti-A, anti-B) are used – serum is
blood plasma without coagulation factors, it contains the relevant agglutinins
̶Mix the antibodies with a drop of blood (sera must not mix)
̶A positive result occurs in serum with agglutination (clumping)
-
+
+
anti-A    anti-B    anti-AB

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Fill correct blood groups to results
anti-A anti-B anti-AB
1
anti-A
anti-B
anti-AB
2
anti-A anti-B anti-AB
3
anti-A
anti-B
anti-AB
4
anti-A
anti-B
anti-AB
5
Department of Physiology, Faculty of Medicine, Masaryk university

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Correct results
anti-A anti-B anti-AB
B
anti-A
anti-B
anti-AB
A
anti-A
anti-B
anti-AB
AB
anti-A
anti-B
anti-AB
0
anti-A
anti-B
anti-AB
wrong sera
Department of Physiology, Faculty of Medicine, Masaryk university