Methods of estimating ESR

2. Objectives
• What is ESR?
• Explain the principle of erythrocyte
sedimentation.
• Discuss mechanism of ESR?
• What are the factors affecting ESR?
• What are methods of estimating ESR?
• What is the clinical significance of ESR?
• What are the source of error while doing the
test?

3. ESR
• Erythrocyte- red cell
• Sedimentation- settling
• Rate- amount in a given
time

5. Introduction
• Also k/a ‘sed’ rate
• Non specific test indicative of inflammation
• It is used as an initial screening tool and also
as a follow-up test to monitor therapy and
progression or remission of disease.
• Easy to perform, inexpensive
• Inexpensive making it a widely used screening
test.

6. • Inexpensive making it
widely used screening test
• Measure- how much red cell
will settle in a given time
• Unit-measured in mm/hrs
Why is the ‘hr’
bit important?

7. Principle
When the anti coagulated blood is allowed to
stand vertically, the red cells will settle towards
the bottom of the tube till they form a packed
column in a given interval of time. The process
of sediment6ation is called ESR.

9. Mechanism
• ESR is determined by the interaction between
factors that promote (fibrinogen) and resist
(negative charge of RBCs - that repel each
other) sedimentation.
• Normal RBCs settle slowly as they do not
form rouleaux. Instead, they gently repel each
other due to the negative charge on their
surfaces.
• Rouleaux are stacks of many RBCs that
become heavier and sediment faster.

10. Mechanism
• Plasma proteins, especially
fibrinogen, adhere to the red cell
membranes and neutralize the
surface negative charges,
promoting cell adherence and
rouleaux formation
• The ESR is directly proportional to
the weight of the cell aggregate
and inversely proportional to the
surface area

11. Stages
• Stage of rouleaux formation/aggregation- 10
min
• Stage of sedimentation/settling- 40 min
• Stage of packing- 10min

12. Factors affecting ESR
Physiological factors
• Plasma factors
• Red cell factors
• Rouleaux formation
• Age
• Sex
• Pregnancy
laboratory factors
• Temperature
• Time
• Anticoagulants
• Tube factor
• Tilting of tube
• Vibration
• Sunlight

14. Plasma factors
• Fibrinogen, globulin and cholesterol increase
ESR by decreasing negative charge of RBC.
Negative charge prevent the RBC coming
together
• Plasma albumin retards sedimentation of
RBCs.
• Lecithin- retard ESR

15. Red cell factor
• Number:
– Increased in red cell mass- decrease ESR
– Decreased red cell mass- increased in ESR
• Size:
– Microcyte sediment slowly and macrocyte
sediment rapidly than normocyte

16. • Shape:
– The shape of the erythrocytes can also affect
the ESR.
– Sickle cell retard ESR because abnormal cell
hamper rouleaux formation
– Spherocytic cells also sediment at a slow rate
Red cell factor

17. Rouleaux formation
• When RBC aggregates to
form a rouleaux and settle
down the area is much less
than sum of the area of
constituent corpuscle
• Hence it is important
factor that increase ESR

18. Age
• Infant- low
• Adult- gradually increase
• Old- higher

19. sex
• Women has slightly higher
ESR then men because of
their low PCV.

20. Pregnancy
• The ESR begins to increase about third
the month and return to normal about
four week after delivery.

22. Temperature
• Test should be done at 20-25⁰ C
• Higher temperature cause false high
results due to reduction in plasma
viscosity
• Always bring refrigerated blood at RT
• Rise in every 3⁰ ESR increase by 1mm

23. Time
• Test should be done within two
hours of collection
• EDTA sample should be kept at
4°C if delayed but should be
performed within 6 hours.

24. Anticoagulant
• K2EDTA or tri-sodium citrate should
be used
• Heparin alter cell membrane potential
and should not be used

25. Tube factor
• ESR is greater with longer tube
• Inner diameter should be 2.5mm or
more to overcome capillary
attraction

26. Position of tube
• Tube should be perfectly vertical
• ESR increase as the RBC slide down
along the lower side
• Angle of 3⁰ from vertical can
increase ESR by 30℅

27. Vibration
• Vibration can reduce ESR

28. Hemolysed or clotted sample
• It retard ESR
• Clot trap fibrinogen hence,
no rouleaux formation occur

29. Sunlight
• Direct sunlight on ESR tube increase ESR

30. Other factors
• Drugs such as dextran, methyldopa, oral
contraceptives, penicillamine procainamide,
theophylline, and vitamin A can increase ESR,
while aspirin, cortisone, and quinine may
decrease it.

31. General assumption

32. Clinical significance
Increased ESR
• Pregnancy
• Anemia
• Macrocytosis
• Inflammatory disease
• Acute and chronic infection
• Multiple myeloma
• Rheumatic fever
• Rheumatoid arthritis
• Anemia
• Tuberculosis
• Systemic lupus erythematosus
Decreased ESR
• Hyperviscosity
• Decreased fibrinogen levels
• Polycythemia
• Sickle cell anemia
• Spherocytosis
• Microcytosis

33. Methods of estimating ESR
• Wintrobe’s method
• Westergren method
• Modified Westergren method
• Automated method

34. Wintrobe’s method
Wintrobe tube:
• It is closed at one end
• length- 110 mm long
• Internal bore diameter 2.5mm.
• Tube is calibrated on both sides:
0-10 and 10-0.

35. Requirement
Sample
• EDTA blood
Apparatus required
• Wintrobe tube
• Wintrobe stand
• ESR needle
• Timer
The procedure must be performed within 2 hours of
blood collection.

36. Procedure
• Mix the anti coagulated blood thoroughly.
• Fill the wintrobe tube by using Pasteur pipette
upto mark 0.
• Place the tube vertically in a stand.
• Note the ESR at the end of 1 hour.

37. Normal range
• Male=0-10 mm in 1st hour
• Female=0-20 mm in 1st hour

38. Westergren method
• Simple and widely used method
• Method recommended by ICHS

39. Requirement
Sample
• Trisodium citrated
anticoagulated blood
• 1.6 ml of blood is mixed
with 0.4 ml of TSC
Apparatus
• Westergren tube
• Westergren stand
• Rubber bulb
• Timer

40. Westergren tube
• Length= 30cm
• Internal bore diameter= 2.5mm
• Can hold about 1ml of blood
• Open at both end
• Calibrated from top to bottom: 0-200mm

41. Procedure
• Mix the blood thoroughly.
• Draw blood into the tube up to mark ‘0’ with
the help of rubber bulb.
• Wipe out blood from bottom of the tube.
• Transfer the tube to westergren stand and
stand it vertically.
• Record the time and take reading.

42. Normal value
• Male= 0-5mm in 1st hour
• Female=0-7mm in 1st hour

43. Advantages
• More reliable and gives
accurate result
Disadvantages
• More blood is required
• Difficult to fill the blood in
tube
• PCV cannot be done
• Mouth pipetting may be
hazardous

44. Modified Westergren Method
• EDTA blood is used instead of citrate.
• 2ml of EDTA blood is diluted with 0.5 ml
of 3.8% TSC or 0.85% NaCl
• Undiluted blood gives poor precision.

45. Automated method
• Ves-Matic
• ESR STAT-PLUS
• SEDIMAT
• Zeta Sedimentation

46. VES-MATIC 20 instrument
• It is designed to measure 20 blood
sample.
• Result are comparable to Westergren
method
• results are available in approximately
22 minutes.

47. Blood is collected in a special cuvette
Sample is mixed by instrument
Sample is allowed to stand
at 18⁰ slant from vertical
Opto-electrical sensor
measure ESR
Data are elaborated and
printed
Working principle:

48. ESR STAT PLUS
• It is centrifugation based method.
• Provides results in 5 minutes.

49. Working principle:
Sample is placed in centrifuge
Infrared laser tracks the erythrocyte plasma interface and takes multiple measurements
Linear portion of sedimentation is identified
Software algorithm to determine ESR result

50. SEDIMAT
• The filled Sediplast Westergren pipet is placed
into the SEDIMAT automated ESR reader,
which accelerates sedimentation under
controlled conditions.
• The reader displays the results of each sample
on an LCD display after 15 minutes.
• The results are also stored in memory and can
be printed out.

51. Zeta Sedimentation Ratio
• The ZSR is performed using a special, small-bore
capillary tube that is filled with blood and spun
for 3 to 4 minutes in a special centrifuge called
the Zetafuge
• Centrifuge alternately compacts and disperses
the RBCs under standardized centrifugal force
• Tube is then read on a special reader to obtain a
value called the zetacrit
• It is rapid, corrects for anemia, and requires only
a small blood sample, which is desirable for
pediatric patients

52. Advantages of automated methods
• Save technician time
• Provide increased safety because the
need for sample manipulation is
decreased
• Interface with laboratory information
systems (LIS)
• Use smaller sample volumes
• Provide more rapid results

53. Source of error
• Improper ratio of blood and
anticoagulant.
• Hemolysed sample.
• Clotted blood.
• Presence of air bubbles.
• Error due to sunlight, vibration, small
bore size, dirty and wet tube.
• Delay in performing the test.