CHAPTER 12:

ERYTHROCYTE
SEDIMENTATION RATE
( ESR )
 Objectives
At the end of this chapter, the students 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
 State the principle of the Wintrobe 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
 Perform ESR measurement on a sample using the
Westergren and Wintrobe methods
 List sources of error in ESR determination
 Indicate the normal values and the clinical implications of
ESR determination
 Perform QC in ESR determination
 Outline

 Introduction
 Stages in ESR determination
 Methods of ESR determination
 Clinical significance
 QC in 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 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

 normal ESR can not be taken to exclude the presence of

organic disease
 majority of acute or chronic infections and most
neoplastic and degenerative diseases are associated
with changes in the plasma proteins which lead to an
acceleration of the sedimentation rate.
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 a set time at the end of which the distance from the
top of the column to the interface between the plasma
and the sedimented red cells is recorded and expressed
in mm/unit time.
12.2. Stages in ESR
ESR has 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.

RBCs Rouleaux
 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..

 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. Influence of Plasma Viscosity
 The ESR and plasma viscosity in general increase in
parallel.
 But plasma viscosity may increase to the extent of
masking the rouleaux forming property of the plasma
proteins.

III. 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..
IV. 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
Two basic methods
 Westergreen and Wintrobe methods

12.4.1 The Westergreen Method

 This is ICSH reference method for ESR determination.
Materials
 Westergreen-Katz tube
 an open glass tube with an overall length of 300mm
and bore of 2.5mm.
 The graduated portion measures 200mm.
 Westergreen rack or stand
 30.88 g/l tri-sodium citrate
 a rubber teat or pipet filler
Westergreen rack & tube
Westergreen rack & tube filled with
blood sample
Procedure

1. Venous blood is diluted accurately in the proportion of

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.
2. A clean dry Westergren-Katz pipette is carefully filled
and adjusted to the "0" mark on top.
Cont’d..

3. The pipet is placed in a strictly vertical position in the

Westergren stand
 under room temperature conditions
 not exposed to direct sunlight and
 away from vibrations and draughts
4. Allow it to stand for exactly 1 hour
5. After 1 hour read to the nearest 1mm the height of the
clear plasma above the upper limit of the column of
sedimenting red cells.
Cont’d..

Reporting
 The result is expressed as ESR = X mm/hr

  A poor delineation of the upper layer of red cells,

the so-called ‘stratified sedimentation’, has been
attributed to the presence of many reticulocytes.
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.
Normal Range:
Men: 0-15mm/hr
Women: 0-20mm/hr
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:

 hypovolemia and
 an increase in concentration of  globulin and
fibrinogen.
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

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 required
 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 low and when the disease is in the acute
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
 Improper filling of tubes
 Old specimen (should be performed within 2 hours of
collection)
 Cold agglutinins can cause a falsely elevated ESR
 Clotted and hemolysed samples
Technical tips

 Tube must be completely filled to the zero mark

 Hemolyzed specimen is not accepted
 There should be no air bubble
 Refrigerated specimens must come to room temperature
for 30 minutes prior to testing
 refrigerated sample is used within 24 hours, if the test
can not be performed within 2 hours of sample
collection
 The ESR rack must be on a level surface and free of
vibration
 Strictly follow SOP
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