CHAPTER 12:

ERYTHROCYTE
SEDIMENTATION RATE
( ESR )
Objectives

At the end of this chapter, the student will be able to:

 Define ESR
 Discuss the factors that affect ESR
 Explain the stages in ESR
 State the principle of the Westergren method of ESR
determination
 List the materials required to perform the ESR by
Westergren and Wintrobe methods
Objectives cont’d

 Discuss the advantages and disadvantages of the ESR

determination by Westergren and Wintrobe methods
 Determine the ESR values of a sample using the
Westergren and Wintrobe methods
 Apply QC in ESR determination
 List at least five sources of error in ESR determination
 Indicate the normal values and the clinical implications of
ESR determination
12.1 Introduction

i. Definition
 Erythrocyte sedimentation rate is the rate of fall
(sedimentation) of red cells when an anticoagulated
blood is allowed to stand vertically undisturbed for a
specified period of time, usually 1 hour. The rate is
expressed in mm/hr.
 It is:
 a non specific test
 used as an index of the presence and extent of
inflammation (the so-called 'acute phase response' )
and its response to treatment, e.g., tuberculosis,
rheumatoid arthritis.
ii. Significance of Measuring the ESR

 ESR is a non-specific test. It is raised in a wide range of

infectious, inflammatory, degenerative, and malignant
conditions associated with changes in plasma proteins,
particularly increases in fibrinogen, immunoglobulins, and
C-reactive protein.
 The ESR is also affected by many other factors including
anaemia, pregnancy, haemoglobinopathies,
haemoconcentration and treatment with anti-inflammatory
drugs.
 Normal ESR can not be taken to exclude the presence of
specific disease
iii. Principle

 The ESR is determined by filling a narrow pipette of

predetermined length and bore, with well mixed
anticoagulated blood and placing it in a vertical position
for one hour, at the end of which the distance from the
top of the plasma column to the interface between the
plasma and the sedimented red cells is recorded and
expressed in mm/hour.
12.2 Stages in ESR

Three stages:
i. An initial period of 10 minutes  rouleaux formation
takes place
ii. A period of approximately 40 minutes  settling or
sedimentation occurs at a constant rate, and
iii. A slower rate of fall (last 10 minutes) packing of the
sedimented red cell column occurs.
* The second stage is the most significant phase.
 12.3 Factors Affecting the ESR

I. Effect of Plasma Proteins

 The relationship between plasma proteins and rouleaux

formation is the basis for measurement of ESR as a non-
specific test of inflammation and tissue damage.

 Red cells possess a net negative charge (zeta potential)

and when suspended in normal plasma, rouleaux
formation is minimal and sedimentation is slow.
 Alterations in proportions and concentrations of various
hydrophilic protein fractions of the plasma occur following
tissue injury or in response to inflammation
Cont’d..

 this reduce the zeta potential and increase the rate of

rouleaux formation and the size of the aggregates
thus increasing the rate of sedimentation.
 The ESR shows a linear relationship with the
concentration of fibrinogen and alpha and beta
globulins. In most acute infections and chronic
pathological processes these fractions are increased
thus enhancing the ESR.
 Albumin which tends to counteract rouleaux formation
diminishes in concentration (hypoalbuminemia) in
inflammatory processes further increasing the
sedimentation rate.
Cont’d
II. Effect of Red Cell Factors
 Efficient rouleaux formation depends on normal shape
and size of the red cells.
 Anisocytosis and poikilocytosis will reduce the ability of
the red cells to form large aggregates thus reducing the
sedimentation rate.
Cont’d..
 Anemia, by altering the ratio of red cells to plasma,
encourages rouleaux formation and accelerates
sedimentation.
 Cellular factors may affect sedimentation. Thus in
iron deficiency anemia a reduction in the intrinsic
ability of the red cells to sediment may compensate
for the accelerating effect of an increased proportion
of plasma.
Cont’d..
III. Effect of Mechanical Influences
The conditions under which the ESR is performed may
influence the results.
 Perpendicularity of the sedimentation pipette
 slight deviations from the vertical will increase the rate
of sedimentation. A 3o inclination can increase the
ESR by 30%.
 Vibration
 vibration can reduce the ESR by retarding the rate of
rouleaux formation
 e.g., centrifugation on the same table
Cont’d..

V. Effect of Temperature
 Higher temperatures cause falsely elevated results
 By reduction in plasma viscosity
 Nevertheless, variation in the ambient temperature of
a laboratory is unlikely to be a significant problem
unless the tubes are exposed to direct sunlight.
12.4 Determination of ESR
There are two basic methods
 Westergren and Wintrobe methods

12.4.1 The Westergren Method

 This is ICSH reference method for ESR determination.
Equipment
 Westergren ESR pipette (Westergren-Katz pipette )
 Westergren pipettes measure 300 mm in length and are graduated from 0-200
mm. The diameter should not be less than 2.55 mm.

 Westergren rack (stand)

 Timer capable of timing accurately 1 hour,
 Reagent
 Tri-Sodium citrate, 31 g/l (3.1% w/v) anticoagulant
Store the reagent at 4-80C. Renew if it becomes cloudy.
 a rubber teat or pipette filler
Specimen
 Either venous blood or EDTA anticoagulated blood .If
EDTA blood is used and kept refrigerated at 4-80C,
citrate dilution of the blood and testing can be delayed
for up to 6 hours.
Procedure

1. Pipette 0.4 ml of sodium citrate anticoagulant into a small

container. and add 1.6 ml of venous blood or EDTA
anticoagulated blood and mix well. i.e one volume of
citrate to four volumes of blood.
 The blood may be directly collected into the citrate
solution or an EDTA anticoagulated blood used.
 Mix thoroughly by gentle repeated inversion.
 ESR preparations should preferably be set up within
2 hrs of blood collection, but under extenuating
circumstances may be refrigerated overnight at 4oC
before testing.
 Cont’d..
2. Using a safe suction method, draw the blood to the 0 mark
of the clean dry Westergren pipette, avoiding air bubbles.
3. Check that the ESR stand is positioned firmly. And position
the ESR pipette vertically on the ESR stand
 under room temperature conditions
 not exposed to direct sunlight and
 away from vibrations and draughts
4. Set the timer for 1 hour.
5. After exactly 1 hour, read the level at which the plasma
meets the red cells in mm. The result is expressed as ESR
= X mm per hour or ESR (WESTERGREN 1HR) = X mm.
6. After reading the ESR, remove carefully the pipette and
soak it in sodium hypochlorite 2500 ppm. Cl (0.25%)
disinfectant
Westergreen method Cont’d..

Advantages
 It more reliably reflects the clinical state
 is the most sensitive method for serial study of
chronic diseases, e.g., tuberculosis.
Disadvantages
 Requires a large amount of blood.
 Involves dilution which may be one source of error.

Reference range
Men ……………………….. Up to 10 mm/hour
Women …………………… Up to 15 mm/hour
Elderly ……………………. Up to 20 mm/hour
Cont’d..
 There is a progressive increase with age as there is
decline in plasma albumin concentration
 ESR is increased in pregnancy as there is a
decrease in plasma albumin due to:
 Increased plasma volume and
 an increase in concentration of  globulin and
fibrinogen.
Causes of a significantly raised ESR
 Anaemia due to any cause
 Acute and chronic inflammatory conditions and infections
including:
 HIV disease
 Tuberculosis
 Acute viral hepatitis
 Pelvic inflammatory disease
Reduced ESR
 Sedimentation is falsely low in polycythaemia,
dehydration, dengue hemorrhagic fever, and other
conditions associated with haemoconcentration.
 Abnormally shaped red cells as in sickle cell disease
also lower sedimentation rate.
 Other situation in which sedimentation is reduced is
during treatment with anti-inflammatory drugs.
12.4.2 The Wintrobe Method

 Uses a tube closed at one end, 11cm long with a bore of

2.5mm and having a graduated scale from 0-100mm
and a special Wintrobe rack
Procedure

1. Blood is collected with EDTA in the right proportion.

2. Enough blood to fill the Wintrobe tube (approximately
1ml) is drawn into a Pasteur pipette having a long
stem.
3. The Wintrobe tube is then filled from the bottom up (so
as to exclude any air -bubbles) to the "0" mark.
4. The tube is placed in the Wintrobe rack in exactly
vertical position and the time is noted.
5. At the end of 1hour the ESR is read as the length of
the plasma column above the cells and is expressed
as x mm/hr.
Wintrobe method cont’d
Advantages
 The method is:
 simple

 requires a small amount of blood

 no dilution requied
 With the same preparation, once the ESR has been
read, the hematocrit value can be determined after
centrifugation.
 Microbilirubin determination can be made on supernatant
plasma and smears of buffy coat can be made.
Wintrobe method cont’d
Disadvantages
 Because of the short column, it is only sensitive when
the ESR is high and when the disease is in the chronic
stage.
 Normal Range
 Men: 0-7mm/hr
 Women: 0-15mm/hr
Quality control

 Strictly adhere to SOP (timing, positioning the ESR rack,

etc)!
 Quality control samples are commercially available
Sources of error
 Using the wrong volume of blood to anticoagulant.
 Blood not sufficiently mixed with anticoagulant.
 Clotted and hemolysed samples- Even the smallest
fibrin clot in the sample will invalidate the test result.
 Air bubbles at the top of the column.
 Testing blood samples at the hottest time of the day, or
leaving tests in direct sunlight. Temperatures over 250C
increase sedimentation.
 Using a pipette which is not clean or not dry.
Source of error cont.

 Pipette not positioned vertically. Even slight variations

from the upright increase sedimentation.
 Not checking whether the ESR stand is level on the
bench.
 Placing an ESR stand on the same bench as a
centrifuge where vibration will interfere with
sedimentation.
 Old specimen (should be performed within 2 hours of
collection)
Review questions

1. Define the ESR

2. What is the principle of ESR determination?
3. What are the stages in ESR that occur in a tube filled
with an appropriately diluted sample of blood?
4. List the items required in ESR determination using the
Westergren method.
5. What is the clinical significance of measuring ESR?
6. List at least five sources of error and their remedies in
ESR determination