eRYTHROCYTE SEDIMENTATION RATE BASICSAND QC

1. Nathan Kikku Mubiru
Medical Laboratory Scientist (MLSO)

2.  Introduction
 Stages in ESR determination
 Methods of ESR determination
 Clinical significance
 QC in ESR determination

3. 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.

4.  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.

5.  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.

6. 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

7. 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

8.  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.

9. 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.

10.  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.

11. 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

12. 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.

13. 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

16. 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.

17. 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.

18. 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.

19. 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

20.  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.

21. 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.

22. 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.

23. 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

24.  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

25.  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

26. 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