(ESR)

Introduction To Medical Technology

- Lab 18 -
 Introduction
† The Erythrocyte Sedimentation Rate (ESR) is a
nonspecific measurement used to detect and monitor
an inflammatory response to tissue injury (an acute
phase) in which there is a change in the plasma
concentration of several proteins (termed acute phase
proteins).
† This procedure, very simply, consist of allowing a
specific amount of blood to sit in a vertical position for
a period of time (usually one hour). The distance, in
millimeters, that the red cells fall during this time period
is the erythrocyte sedimentation rate and is reported in
mm/hr.
† The ESR is affected by three factors:
† Erythrocytes,
† Plasma composition,
† Mechanical/technical factors.
 I. Erythrocytes
† A factor of chief importance in determining the
distance that the RBCs falls is the size or mass
of the falling particle. The larger the particle, the
faster its rate of fall.
† In normal blood, the RBCs remain more or less
separated. They are negatively charged and,
therefore repel each other.
† In certain diseases, however, plasma protein
concentration may be altered, causing a
reduction in the negative charge of the RBCs and
consequent formation of rouleaux. This leads to
a larger mass and an increased sedimentation
velocity.
† Agglutination of RBCs due to change in the
erythrocyte surface also leads to an increased
RBC mass and a more rapid sedimentation rate.
† Macrocytes tend to settle more rapidly than
microcytes.
† Anisocytosis and poikilocytosis reduce the ability of
RBCs to form large aggregates and there by tend to
falsely lower ESR.
 RBCs show an alteration in shape, such as sickle
cells and spherocytes, are unable to aggregate or
form rouleaux and the sedimentation rate is
decreased.
† In severe anemia, the ESR is markedly elevated.
 The concentration of RBCs is decreased,
aggregation and rouleaux formation are increased,
and they therefore, settle out more easily and
rapidly.
† In polycythemia, in which the RBCs count is high, the
ESR is generally normal.
II. Plasma composition
† The plasma composition is the most
important factor determining the ESR.
Rouleaux and aggregation of the RBCs are
controlled primarily by the levels of acute
phase proteins (most notable fibrinogen, -1
globulin, and -2 globulin); increasing these
three plasma protein levels are increased in
the plasma.
† As the concentration of protein increases so
does the viscosity of the plasma. Although an
increased plasma viscosity will tend to inhibit
the fall of the RBCs, the increase in plasma
proteins are generally those which cause
rouleaux and aggregation of the RBCs, which
affects the ESR more greatly than does the
increased plasma viscosity. Increased
concentrations of albumin will tend to lower
the ESR.
III. Mechanical/Technical
factors
† It is important that the ESR tube be exactly
perpendicular. A tilt of 300 can cause errors up
to 30%.
† Also, the rack holding the tubes should not be
subject to any movement or vibration.
† Minor, everyday variations in room temperature
do not significantly affect the ESR. With large
changes in temperature, however, the
sedimentation rate increases as the
temperature increases.
† The length and inner diameter of the ESR tube
also affect the final test results. ESR tubes with
a narrower than standard bore will generally
yield lower sedimentation rates.
 Significance of the ESR
† The ESR represents a nonspecific response
to tissue damage and inflammation and
denotes the presence of disease, but not its
severity.
† It primarily reflects changes in the plasma
proteins that accompany most the acute and
chronic infections, tumors, and degenerative
diseases. It may be used to follow the
progress of certain diseases such as
tuberculosis and rheumatoid arthritis.
 An elevated ESR may be found in:
1. Pregnancy (after the third 7. Acute hepatitis.
month). 8. Menstruation.
2. Acute and chronic infections. 9. Tuberculosis.
3. Rheumatic fever. 10. Hypothyroidism.
4. Rheumatoid arthritis. 11. Hyperthyroidism.
5. Myocardial infection.
6. Nephrosis.

 Adults over 60 years of age frequently have a

slightly higher ESR value due primarily to
decreased concentrations of plasma albumin.
A decreased ESR will be present in:

1. Polycythemia.
2. Congestive heart failure.
3. Hypo-fibrinogenemia.
4. The presence of red blood cell
abnormalities (poikilocytosis,
spherocytes, and sickle cells).
 I. Westergren method
Principle
† Whole blood (4 volumes) anti-coagulated with 0.109M
tri-sodium citrate (1 volume), or EDTA anti-coagulated
blood (4 volumes) diluted with 0.85% sodium chloride (1
volume) is mixed, placed in a Westergren pipette, and
allowed to stand for exactly 1 hour in a vertical position.
The number of millimeters the red blood cells fall during
this timed period constitutes the ESR.
† The normal values for the modified Westergren ESR are
 0 to 15 mm/hour for women,
 0 to 10mm/hour for men,
 0 to 10mm/hour for children.
 Reagents and Equipment
1. Disposable Westergren pipette calibrated in millimeters.
The National Committee for clinical laboratory standards has set
specific dimensions for the pipettes to be used:

 Length = 300.5mm (+/-0.5mm) (in

order to fit into standard racks)
 External diameter = 5.5mm (+/-
0.5mm)
 Internal bore = 2.65mm (=/-
0.15mm)
 Graduated scale on pipette =
200mm (+/- 0.35mm) (Graduated
lines of uniform thickness 0.2mm)
2. Westergren pipette rack. All racks should
contain a leveling bulb in order to ensure that
the position of tubes is vertical (+/-2o).
3. Leveling plate for holding the Westergren rack
4. Timer.
 Specimen

† Whole blood (4 volumes) diluted with 0.109

M tri-sodium citrate (1 volume). Alternatively,
3ml whole blood anti-coagulated with EDTA.
(If this specimen type is used the whole
blood [2.0ml] must be diluted with 0.85% w/v
sodium chloride [0.5ml] prior testing).
Procedures
1. Mix the diluted blood for at least 2 minutes on a
rotator. (The blood should be at room
temperature). Check the tube for clots using two
applicator sticks.
2. Make certain the Westergren ESR rack is exactly
level.
3. Fill the Westergren pipette to exactly the 0 mark.
Making certain there are no air bubbles in the
blood.
4. Place the pipette in the rack. Be certain the pipette
fits snugly and evenly into the grooves provided.
5. Allow the pipette to stand for exactly 60 minutes.
6. At the end of 60 minutes records the number of
millimeters the red blood cells have fallen. This
result is the erythrocyte sedimentation rate in
millimeters/hour
II. Wintrobe and Landsberg
method
Principle
† Well-mixed, whole blood is placed in a
Wintrobe tube and allowed to stand for 1
hour. The number of millimeters the red
blood cells fall during time constitutes the
ESR. In the Wintrobe and Landsberg
method, normal values for women are0 to 20
mm/hour and 0 to 9 mm/hour for men.
Reagent and equipment
† Wintrobe tube, calibrated in
millimeters
† Wintrobe pipette rack.
† Disposable capillary pipette.
† Applicator sticks

Specimen
† Whole blood, 1 ml, using EDTA as the
anticoagulant.
Procedure

1. Mix the whole blood for at least 2 minutes

on a rotator. (Make certain the blood is at
room temperature). Check the tube for
clots using tow applicator sticks.
2. With a capillary pipette, fill the Wintrobe
tube to the 0 mark.
3. Place the tube in an exactly vertical
position in the rack. Time for 60 minutes.
4. At the end of 60 minutes, record the level
of the erythrocyte column. This result is
the erythrocyte sedimentation rate in
millimeters/hour.
1.
Discussion
The sedimentation of red blood cells takes
place in three stages:
 In the first 10 minutes (Lag Phase) rouleaux
formation occurs and the sedimentation rate is
slight.
 Sedimentation then occurs for a period of
approximately 40 minutes at a more rapid and
constant rate (Decantation).
 During the last 10 minutes, the sedimentation
rate is slow because of the accumulation of
RBCs in bottom of the tube. The longer the
tube the longer the stage two will last, and
therefore the higher the ESR result
(Stationary).
2. Although care may be taken in filling the
sedimentation tube to the 0 mark, occasionally
the upper level of the blood may only reach the 1
or 2 mm mark. In such case, care should be
taken when making the final reading. Subtract
the 1 or 2 mm from the final result.
 For example,
 If the ESR tube is filled to the 2 mm mark and the
RBCs fall to the 18mm mark, the ESR is reported as
16mm/hour.
 If the level of blood falls below the 5 mm mark, the
test should be repeated to ensure that valid results
are obtained.
 If the upper level of the blood is below 0 due to
leakage of the blood from the bottom of the tube
(Westergren method) the test must be repeated from
step 1. leakage of blood from the bottom of the tube
invalidates the test result.
3. All the sedimentation racks should be equipped
with leveling screws and a spirit level.
 4. Mechanical sources of error:
A. If the concentration of EDTA is greater than
recommended, the ESR will be falsely low as a result of
inhibiting rouleaux.
B. The anticoagulants sodium or potassium oxalate and
heparin cause the RBCs to shrink and the ESR will be
falsely high.
C. If the ESR stands for more than 60 minutes, the results
will be falsely elevated. If the test is timed for less than
60 minutes, invalidly low values are obtained.
D. A marked increase (or decrease) in room temperature
leads to increased (or decreased) ESR result.
E. Tilting of the ESR tube increases the ESR.
F. Bubbles in blood cause invalid results.
G. Fibrin clots present in the blood invalidate the test results.

5. The ESR should be set up within 2 hours of blood

collection. If EDTA is used as the anticoagulant, the test
may be set up within 6 hours if the blood has been
refrigerated.
6. The Wintrobe ESR technique is thought to be more
sensitive when the ESR is low, whereas the Westergren
procedure more accurately reflects the patient’s disorder
when the results are high. The Westergren method has
been chosen as the standard method by the International
Committee for Standardization in Hematology.
7. When reading the ESR results on bloods with an
extremely high WBCs or platelet count, the buffy coat
should be excluded from the reading.
8. If there is poor separation of the RBCs and plasma layers
this may be due to an increased number of reticulocytes
and has been termed “stratified sedimentation”.
9. The internal bore and the length of the graduated scale
on the Westergren pipette are critical measurements.
 Disposable ESR

† Commercial kits are now available for a

disposable ESR test. Several kits include
safety caps for the pipettes that allow the blood
to precisely fill to the zero mark. This makes
the pipette a closed system and eliminates the
error involved with manually setting the blood
at the zero mark.