ISTINYE UNIVERSITY MEDICAL FACULTY

PHYSIOLOGY LAB NOTES

BLOOD SAMPLING

Capillary blood sampling

Capillary blood is obtained for some experiments in which a small amount of blood sample is n
eeded. Some of these experiments are:

 Determination of hematocrit value

 Measurement of hemoglobin value
 Erythrocyte / leukocyte count
 Peripheral smear
 Blood group determination
 Determination of coagulation / bleeding time
 Blood glucose, urea, bilirubin, pH, and similar tests
 To carry out neonatal screening test in newborn

Capillary blood samples are obtained from the 3rd or the 4th fingertips or earlobe of adults and from
toes or heels of babies.

Tools and Equipment: 70 % alcohol, cotton, sterile lancet.

Procedure

1- Clean the proposed puncture site with a alcohol soaked-cotton. Allow the area to air dry.
Open the safety lancet package, as shown in figure
2- Perform skin-puncture with sterile lancet. In order to achieve enough blood and to prevent
the need to repeat the puncture, the lancet ought to be held in a perpendicular position to
the fingertip and hitting with one quick, continuous and deliberate stroke by a wrist
movement
3- The color of the fingertip region where blood samples is collected should be normal and the
area shouldn’t be a wounded, edematous or inflamed.
4- After pricking the fingertip with a lancet, fingertip ought to be held straight down, so this
position facilitates blood sampling due to gravity.
5- Wipe away the first drop of blood, since the first drop could contain tissue fluid or debris.
6- Avoid applying too much pressure to the area from which capillary blood sample is being
taken (for example, squeezing the fingertip too much), since extreme pressure provokes
rushing of extracellular tissue fluid into the capillaries, resulting in more dilute blood samples
with tissue fluid (plasma) and also increases the possibility of hemolysis. This situation affects
accuracy of the analysis.
7- After the blood collection procedure is complete, apply firm pressure to the puncture area to
stop the bleeding.
MEASUREMENT OF HEMATOCRIT VALUE

Hematocrit is the ratio of the blood cell in blood to the total blood volume. Measurement of the
hematocrit is based upon separation of blood cells from the plasm by centrifugation after the
coagulation of blood is prevented.

The ratio of the blood cells to the total blood volume gives hematocrit value in percentage terms.
The volume of leukocytes and thrombocytes in precipitated cellular blood cells compared to that of
erythrocytes has a negligible value, hematocrit value can be defined as the ratio of erythrocyte
volume to the total blood volume.

Hematocrit values can be assessed by microhematocrit method. In a healthy individual, hematocrit

value is;

 %47 ± 5 in men,
 %42 ± 5 in women.

Normal values of hematocrit actually depend on age and gender. In people living at high altitudes,
hematocrit level is also higher than normal. An individual who has an abnormally low hematocrit is
referred to as being anemic. In other words, anemia indicates an abnormally low hematocrit level,
but polycythemia refers to an abnormally high hematocrit level.

Similarly, if the body senses low oxygen levels, number of red blood cells would increase in an effort
to increase the amount of oxygen in the blood in particularly lung or heart diseases.

The hematocrit values are relatively high in dehydration or burning. Because the liquid part of blood
can be compensated more rapidly as compared with erythrocytes after hemorrhage. But in fact,
hematocrit value may be lower than normal.

Tools and equipment: Heparinized capillary tube (1x70 mm), stove, micro hematocrit centrifuge,
hematocrit reader card, lancet, cotton and alcohol.

Procedure

1- Wipe the fingertip with an alcohol soaked-cotton.

 2- After pricking the fingertip with a lancet, the colored end of the heparinized capillary tube
held horizontal to the ground is touched to the blood drop in the fingertip. The capillary tube
is filled with blood through the capillary effect. Make sure that the tube is filled up to the 2/3
or ¾ mark and then the same end of the capillary tube is sealed with stove.
3- The capillary tube is placed into the groove in the table of the microcentrifuge device in a
position its sealed end remains outward. Another capillary tube containing other volunteer’s
blood is placed into opposite groove for balance. Which capillary tubes are placed into which
groove should be written down.
4- The capillary tubes are centrifuged at 10.000 spins per minute for 5 minutes.
5- At the end of the centrifugation, hematocrit capillary pipette will be separated into three
compartments. The erythrocytes form a red band in the undermost layer. Over this layer,
there is a very thin cloudlike layer composed of lymphocytes and thrombocytes. At the very
top part of the capillary there is a transparent plasma layer.
6- The upper limit of plasma is taken as 100 and the lower limit of erythrocytes is considered as
0 point. In this way, the value of erythrocytes can be calculated.

Problems that may be encountered during application:

 The presence of air bubbles in the tube.

 If the open end of the tube cannot be sealed with paste properly, blood can leak out during
centrifugation.
 If the tubes cannot be settled down on centrifugation device symmetrically, the tubes can be
broken down.
ERYTHROCYTE SEDIMENTATION RATE (ESR)

After coagulation of the blood has been prevented by using an anticoagulant substance, settling rate
of erythrocytes in a tube over a given period of time is called sedimentation rate. It is expressed in
terms millimeters per hour (mm/hour).

After the application of anticoagulation substance erythrocytes tend to sink to the bottom after a
while. Plasma membranes of erythrocyte are negatively charged (zeta potential) because of sialic
acid present in the membrane, which prevents sedimentation of erythrocytes. There is a balance
between prosedimentation factors, mainly fibrinogen, and antisedimentation factors, namely the
negative charge of the erythrocytes. If there is an inflammation, the increased fibrinogen level in the
blood due to inflammation causes red blood cells to stick to each other.

The sedimentation of erythrocytes can increase in some conditions including infectious diseases,
autoimmune disease, rheumatoid arthritis, cancer etc. So ESR indicates the presence of inflammation
associatedwith these conditions. ESR help doctors diagnose or monitor the progress of an
inflammatory disease.

Several factors can have an effect on sedimentation rate of the erythrocytes. These factors can be
related to erythrocytes, plasm, mechanical or technical sources.

Factors related to Erythrocytes

 As the diameter and weight of erythrocytes are increases (for example in the case of
macrocytosis), ESR increases. But note that in megaloblastic anemia, a kind of macrocytic
anemia, ESR decreases.
 The conditions in which rouleaux formation develops (chronic liver diseases,
macroglobulinemia, and myeloma) increase ESR.
 In anemias, the concentration of erythrocytes decreases but ESR increases.

Factors related to Plasma

 In the cases that some plasma proteins such as fibrinogen, alpha 1-globulin, and alpha 2-
globulin accelerate the aggregation of erythrocytes, so ESR increases.

Mechanical and Technical Factors

 In high room temperature, ESR increases

 Tilting the pipette results in increased ESR
 Keeping the blood before the test more than one hour decreases ESR
 Excess use of anticoagulant substance in blood decreases ESR
 When the diameter of the tube exceeds 2 mm, ESR decreases.

Normal ranges of ESR values

 0-20 mm / hour in women

 0-15 mm / hour in men
 0-10 mm / hour in children
Tools and Equipment:

 Syringe ( 2-5 ml ),
 %3.8 of Sodium Citrate solution,
 Sedimentation tubes and pipettes ( Westergreen ),
 tube ( 13x100 mm )
 chronometer.

Procedure:

1- Venous blood sample is obtained.

2- After the syringe is taken out the
vessel, the blood is poured slowly so
as not to hemolyze into the
sedimentation tube containing 0.2 ml
of %3.8 of Sodium Citrate up to the
marked line. (Sodium Citrate is an
anticoagulant, chelating calcium ions)
3- Shake the tube properly to mix the
blood and Sodium Citrate.
4- Then, the Westergreen Pipette, which
is about 20 cm in length and labeled
up to 170 mm, 2.5 mm in inner
diameter and takes about 1.2 ml of
blood, is placed carefully into the
tube. The blood should reach up to
the zero line of the Westergreen
pipette.
5- Press the button of a chronometer
immediately after the pipette is
placed inside the sedimentation tube.
At the end of the half hour, one hour,
two hours and the twenty forth
hours, sedimentation levels of the
blood cells are noted down. Usually,
the value at the end of the first hour is taken as ESR value. The ESR is the value where the
plasma separates from the blood cells.
 Problems that may be encountered during the practical:

 If the pipette is not placed properly inside the Sodium Citrate containing tube.
 If the blood is not risen to the zero point after the pipette has been placed inside the
tube
 The pipette might be broken if the pipette has been sunk into the tube rigorously.

DETERMINATION OF BLOOD TYPES

Blood groups are based on the presence or absence of inherited antigens (agglutinogen) on the
surface of red blood cells. These antigenic substances are glycoproteins in nature and the
carbohydrate is the major component that determine blood type. Whenever red blood cells that
have one particular blood type antigen are mixed with the serum that contain antibodies against that
particular antigen, the antibodies as an immune system defense mechanism attack to the antigen,
resulting in the formation of clumps of red blood cells (agglutination). Blood typing is done by
determining whether clustering of erythrocytes are present. The most commonly used human blood
grouping system is the ABO system which was developed in the 1901 by Landsteiner.

Tools and Equipment:

 Anti – A, Anti – B, Anti Rh (Anti D) test serums,

 lancet,
 alcohol,
 cotton
 microscope slide
 pin

Procedure

1- One drop of blood is added to each, one end, the other end and the middle of a clean slide
(there should be enough distance between the drops).
2- A drop of Anti-A test serum is added to the first drop, a drop of Anti-B test serum is added to
the middle drop and a drop of Anti-D test serum is added to the last drop in order. Above all,
which antibodies has been dropped to which blood drop should be kept track.
3- After the antibodies have been added, the blood drops are mixed with the serum properly
using corner of another microscope slide or a pin. During these mixing processes, care must
be taken to prevent the mixing of the drops.
4- Check is if agglutination occurs.
Problems may be encountered during the practical

 The Anti – A, Anti – B, Anti – D serums may not work or confusing the order of the anti-
serums.
 The blood drops can touch to each other while mixing the drops.
 It may take long time for the serums to show their effects.
 The blood drop may be too small or the blood is clotted due to waiting too long