Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
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.
Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
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
Name number Study group
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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)
Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
Results
Red blood cell count
Describe the method:
…………………………………………………………………………………………………
…………………………………………………………………………………………………
…………………………………………………………………………………………………
…………………………………………………………………………………………………
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
Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
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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Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
Protocol
Determination of blood groups by slide
method
Methods
Equipment:
Standard sera of group A (anti-B, i.e. beta), group B (anti-A, i.e. alpha) and group 0 (anti-A
and anti-B, i.e. alpha and beta), slides, dropper
Procedure:
1. Drop each serum on the slide. Be careful not to mix them!
2. Using a dropper with a fine tip, take a drop of blood from the blood sample.
3. The corner of another slide is put into the blood drop and a small amount of blood is
transferred to one of the sera and mixed by the same corner. In the same way, but
using always another corner of the slide, transfer blood to the remaining test sera and
mix the samples carefully.
4. Agglutination may be speeded up (2–10 min) by carefully rocking the slide to all four
sides. The agglutination appears as visible flakes floating in the transparent serum.
The result may be tested by microscopic observation: in the case of a positive reaction,
the rod cells will form larger clusters.
Note:
Do not confuse the terms agglutination and coagulation, as these designate totally
different physical and/or chemical events. Unfortunately, sometimes coagulation does appear
in the test drop, usually when too much blood is added and mixed only slightly or not at all.
To prevent this, the ratio of blood to test serum should be around 1:10.
Name number Study group
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Results
Into the scheme below draw the reaction observed in your conducted experiment.
Blood 1
Blood group determined in your experiment:
This particular blood group is found in …………% of population.
Based on Mendel’s laws suggest all the possible combinations of the parent’s blood groups.
…………….. ……………. .……………..……………. .……………..
Suggest combinations of parent’s blood groups unable to produce your patient’s blood group.
…………….. ……………. .……………..……………. .……………..
Blood 2
Blood group determined in your experiment:
This particular blood group is found in …………% of population.
Based on Mendel’s laws suggest all the possible combinations of the parent’s blood groups.
…………….. ……………. .……………..……………. .……………..
Suggest combinations of parent’s blood groups unable to produce your patient’s blood group.
…………….. ……………. .……………..……………. .……………..
Conclusion
Propose possible blood groups of acceptors compatible for receiving your patient’s blood
products – blood plasma and packed RBCs.
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Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
Protocol
Erythrocyte sedimentation rate
Methods
Equipment: Stand with a set of sedimentation pipettes, rubber cups, sucking rubber
cylinders, six samples of blood:
• anti-coagulated human blood with full plasma
• anti-coagulated bovine blood with full plasma
• anti-coagulated horse blood with full plasma
• anti-coagulated human blood, the plasma of which is totally replaced by physiological
solution
• anti-coagulated horse blood, the plasma of which is totally replaced by physiological
solution
• anti-coagulated human blood with low R.B.C. (Ht = 0.29)
Procedure:
1. Mix well the anti-coagulated undiluted blood (use gloves!) and pour 2–3 ml into the
first rubber cup which is placed in the stand in such way that the tip of the
sedimentation pipette is exactly in the centre.
2. The rubber cylinder is put on the upper end of the pipette and used to suck up the
blood. By slightly pressing the cylinder, a small amount of air is expelled from the
pipette, the lower end of which is then immersed into the cup filled with blood.
Release gradually the cylinder to suck blood up to the mark 0 (height 200 mm). In that
moment, press the pipette down towards the bottom of the rubber cup. Then, with the
hand which was used to suck up the blood, fix the pipette in this position by tightening
its screw. In the same way, the remaining two pipettes are filled with the other
samples.
3. Read the sedimentation values every 15–20 minutes so that you obtain at least 3
values (in clinical practice, values after 1 and 2 hours are recorded).
Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
Results
Graph of erythrocyte sedimentation rate (choose different colours for each specimen):
Time (min) 15 30 45 60 75 90 105 120
human blood with full
plasma
Human erythrocytes
with physiol. solution
bovine blood with full
plasma
horse blood with full
plasma
human blood with low
R.B.C.
horse erythrocytes with
physiol. solution
List all the factors influencing the erythrocyte sedimentation rate:
………………………………………………………………………………………………
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Conclusion
Explain the differences between the blood samples you worked with.
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Name number Study group
This text was supported by project FRMU no. MUNI/FR/1552/2015 (Faculty of Medicine, Masaryk University).
Protocol
Osmotic fragility test
Methods
Equipment: Stand with 13 test tubes, solution of 1% NaCl, distilled water, physiological
solution (0.9% NaCl), 2 graduated pipettes (10 ml), dropper, anti-coagulated human blood,
gloves.
Procedure:
1. Twelve test tubes are filled, by using two pipettes, with decreasing amounts of 1%
solution of NaCl and increasing amounts of distilled water according to the table
below. The last test tube is filled with the physiological solution (0.9% NaCl). Always
use a clean pipette for each test tube.
Tube number 1 2 3 4 5 6 7 8 9 10 11 12
NaCl 1%
(ml)
6.3 6.0 5.7 5.4 5.1 4.8 4.5 4.2 3.9 3.6 3.3 3.0
H2O
(ml)
3.7 4.0 4.3 4.6 4.9 5.2 5.5 5.8 6.1 6.4 6.7 7.0
% NaCl 0.63 0.60 0.57 0.54 0.51 0.48 0.45 0.42 0.39 0.36 0.33 0.30
Table for preparation on NaCl solutions of decreasing osmotic pressure
2. Each test tube is carefully mixed.
3. Two drops of blood are placed into each tube and the content is again carefully mixed.
After this, do not move the stand or the test tubes so as not to interrupt the
sedimentation of non-haemolysed red cells.
4. After approx. 2 hours (min. after 30 minutes) of standing at room temperature,
estimate the range of haemolysis by examining the colour of the supernatant solution
above the sediment cells and the intensity of turbidity of the cell suspension. The tube
with the normal physiological solution serves as the control since blood taken some
time ago may already be partially haemolysed.
In the lowest concentrations of NaCl a complete haemolysis occurs, i.e. all red cells
disintegrate. The content of the tube is quite pellucid (a text can read through the tube). At a
certain concentration, however, the haemolysis is not complete, as a portion of the blood cells
(i.e. those with the highest resistance) did not haemolyse, which appears as a slight turbidity
in the lower part of the tube – incomplete haemolysis. The lowest concentration where a small
fraction of cells remained non-haemolysed designates the s.-c. maximal resistance. On the
other end of the concentration scale one finds a tube where the haemolysis is hardly
distinguishable: the solution above the sediment is only slightly coloured by haemoglobin
Name number Study group
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released from the least resistant cells. The concentration where osmotic haemolysis just
begins to appear is designated as the minimal resistance. When non-haemolysed erythrocytes
form a non-transparent sediment on the bottom of the test tube with a yellowish fully
transparent NaCl solution above it, such a test is designated as no haemolysis.
The osmotic resistance range is the difference between the maximal and minimal resistance
value.
Results
 Minimal osmotic resistance was observed in ………%NaCl (physiological: 0.4 - 0.44 %)
 Maximal osmotic resistance was observed in………%NaCl (physiological: 0.3 - 0.34 %)
 Mark osmotic resistance range.
Conclusion
Note down all the types of haemolysis known to you (there are 6 types) and explain their
mechanism.
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