ERYTHROCYTE SEDIMENTATION RATE: METHOD, PRINCIPLE AND INTERPRETATION.

1. ESR – PRINCIPLE,
METHODS AND
INTERPRETATION

2. DEFINITION
• The rate at which erythrocytes settle down at
bottom of the tube at 1st one hour is regarded as
Erythrocyte Sedimentation Rate.
• ESR is a commonly used nonspecific test in
routine clinical practice.

3. CHARACTERISTICS
• Indicative of inflammation.
• Used as initial screening tool.
• Follow up test to monitor therapy and progression/remission of disease.
• Easy to perform.
• Inexpensive.
• Unit – measured in mm/hr.

4. CLINICAL SIGNIFICANCE OF
ESR
• To follow the course of disease.
• To establish the prognosis in certain chronic
disease.
• To support the diagnosis.

5. ZETA POTENTIAL
• Measure of magnitude of
electrostatic charge or
repulsion/attraction between
particles.
• Zeta potential results from
negatively charged sialic acid
groups on RBC’s membrane.

6. MECHANISM.
Normally, RBCs
settle down
slowly as they do
not form
rouleaux. Instead,
they gently repel
each other.
.
ESR is
determined by the
interaction
between factors
that promote
(fibrinogen) and
factors that resist
(negative charge
of RBC)
sedimentation.
.
Plasma proteins,
especially
fibrinogen,
adhere to the red
cell membranes
and neutralize the
surface negative
charge promoting
cell adherence
and rouleaux
formation.

7. STAGES OF ERYTHROCYTE
SEDIMENTATION
Stage of rouleaux formation/ lag phase - 10 minutes
Stage of sedimentation/setting – 40 minutes.
Stage of packing – 10 minutes

8. FACTORS THAT INCREASE ESR
• Old age
• Female
• Pregnancy
Physiological
Factors
• Anemia
• Elevated fibrinogen levels
• Infection
• Inflammation
• Malignancy
Pathological
Factors
• Technical factors
• Dilution problems
• Increased temperature of specimen
• Tilted ESR tube
Mechanical
Factors

9. FACTORS THAT DECREASE ESR
Pathological Factors
• Protein abnormalities
• Hypofibrogenemia
• Hypogammaglobulinemia
• Dysproteinemia
• Extreme leucocytosis
• Polycythemia
• RBC abnormalities-
Spherocytosis,
Acanthocytosis, Microcytosis
Mechanical Factors
• Technical factors
• Dilution problems
• Inadequate mixing
• Clotted blood sample
• Short ESR tube
• Vibrations during testing

10. METHODS
Westergren method.
Wintrobe method.
Micro- ESR method
Automated methods.

11. WESTERGREN METHOD
International reference method.
REQUIREMENTS:
1. Westergren ESR pipette-
• Straight glass pipette.
• 300 mm in length.
• 0-200 marking from top to bottom- only
in lower 2/3rd of tube.
• Diameter 2.55mm.

12. 2. Westergren stand- holds tube in vertical,
motionless position.
3. Anticoagulant- Trisodium Citrate Dihydrate
is anticoagulant of choice.
Composition –
• Trisodium Citrate Dihydrate- 32 gm
• Distilled water – 1000 ml

13. PROCEDURE
• 2ml of whole blood + 0.5ml of sodium citrate (4:1)
• Westergren pipette is filled to 0 mark and placed vertical
in rack at room temperature without vibration or
exposure to sunlight.
• After 60 min, distance from 0 mark to top of
column of red cells is recorded in mm as ESR
value.

14. MODIFIED WESTERGREN
METHOD
• Only modification-It uses EDTA as anticoagulant
rather than with citrate.
• 2ml of well mixed EDTA blood is diluted with
0.5ml of 3.8% sodium citrate or 0.85% of sodium
chloride.

15. REFERENCE RANGE
• Reference range-MEN WOMEN
Below 50 years of age 0-15mm/ hour 0-20mm/ hour
Above 50 years of age 0-20mm/ hour 0-30mm/ hour
Above 85 years of age 0-30mm/ hour 0-42mm/ hour
Newborns 0-2mm/ hour
Children to puberty 0-13mm/ hour
Henry’s clinical diagnosis and management by laboratory methods

16. PRECAUTIONS
• Wash the pipette as early as possible.
• Rinse in deionized water and dry in incubator
between 40-50℃.

17. WINTROBE’S METHOD
It can be used for both ESR and PCV.
REQUIREMENTS:
• Wintrobe’s tube:
 110 mm in length.
 3 mm in diameter.
 Two markings: 1st marking for ESR
(above downwards), 2nd marking
for PCV (below upwards).

18. • Vertical stand: holds tube in
vertical and motionless
position.
• Anticoagulant: EDTA or
Double oxalate.
• Pasteur pipette: To fill
Wintrobe tube (2ml syringe
with needle can also be used).

19. PROCEDURE
• Fill the Wintrobe tube to 0 mark by using
Pasteur pipette or by using syringe.
• Place the tube in exact vertical position in
the stand.
• At the end of 1 hour note the level of
erythrocyte level in terms of mm.

20. • Reference range
RANGE
Males 0-9mm/ hour
Females 0-20mm/ hour
Children 0-13mm/ hour

21. PRECAUTIONS
• Wash the tubes under running tap water by
introducing a thick wire in tube repeatedly to
remove packed cells completely.
• Dry tubes in incubator (40-600 C).
• In case of infants or insufficient blood quantity use
Micro-Sedimentation (Landau) method.

22. Micro –ESR method
• Landau-Adams micro sedimentation
method.
REQUIREMENTS:
• Fasting capillary blood (finger tip,
heel or toe).
• 5.0 g/dl sodium citrate solution.
• Landau pipette graduated from 0-
50mm (resembles RBC pipette).
• Landau pipette stand.
• Suction device.

23. PROCEDURE
• Attach the landau pipette to suction device.
• Draw 5.0 g/dl citrate upto the first line on stem.
• Now draw blood by suction device upto second mark on stem.
• Mix citrate solution and blood in bulb of pipette.
• Set the upper level of mixture to 0 mm mark at top.

24. • Normal values: RANGE
Males 0-5mm/ hour
Females 0-8mm/ hour
Newborn 0-2mm/ hour

25. Corrected ESR
• To eliminate the influence of anemia on ESR.
• Corrected according to the volume of Redcells.
• For correction we use Fabry’s formula.
• Fabry’s formula:
Corrected ESR = Measured ESR x 15/ (55-Hct)

26. Automated methods
• Ves-matic 20
• ESR-Stat plus
• Sedi-mat 15

27. VES-MATIC 20
Bench top automated analyzer.
Blood is collected in special cuvets.
Sample are then left to sediment for certain
period.
18 degree slant of tubes with respect to vertical
axis causes acceleration of sedimentation.
This slant allow results comparable with those
of Westergren at first hour in 25 minutes only.

28. VES-MATIC 20
Opto electrical sensor
measure ESR.

29. ESR STAT-PLUS
• It is a Centrifugation basedmethod.
• Provides results in 5minutes.
• 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.

30. SEDI-MAT 15
• Filled sedimat Westergren pipette
is placed in SEDIMAT 15 which
accelerates sedimentation under
controlled condition.
• Based on the ability of blood to
block the transmission of infra
red rays.
• The reader displays result on
LCD screen after 15 minutes.

31. ZETA SEDIMENTATION RATE
• More accurate data than ESR.
• Not affected by anemia.
• Small amount of blood for
testing.
• Provides fastest result.
• Requires a Zetafuge.
• Four 45 second cycles of
dispersion and compaction in
capillary tubes.

32. PROCEDURE
• Fill a capillary tube with blood sample.
• Place the capillary tube vertically into zetafuge.
• Spin four cycles of 45 seconds.
• Read the capillary tube like a standard haematocrit tube.
• Result are called Zetacrit.
• Zeta sedimentation ratio = (True Hct/Zetacrit) X100

33. Values of ZSR
• Normal:51-54%
• Mildly elevated:55-59%
• Moderately elevated:60-64%
• Markedly elevated:>64%

34. ADVANTAGES OF
AUTOMATED METHODS
 Save technician time.
 Requires small sample volume.
 Generate more rapid results.

35. SOURCES OF ERROR
• Haemolysed bloodsample.
• Clottedblood.
• Presence of airbubble.
• Error due to sunlight, vibration, small bore size,
dirty and wet tube.
• Delay or wrong method in performing thetest.

36. INTERPRETATION
• In conclusion, conditions with very high ESR (more than 100mm/ hour)
are:
1. Multiple myeloma
2. Connective tissue disorders - SLE, RA and other autoimmune diseases
3. Tuberculosis
4. Temporal arteritis
5. Severe anemia
6. Malignancies
*ESR has little diagnostic value in these disorders but can be useful in monitoring disease activity.

37. INTERPRETATION
• Conditions with low ESR:
1. Polycythemia
2. Severe Leukocytosis
3. Sickle cell anemia
4. Hereditary spherocytosis
5. Hypofibrinogenemia
6. Low plasma protein due to
liver or kidney disease.

38. LIMITATIONS OF ESR
• ESR is a non specific phenomenon and reflects
only change in plasma protein pattern and the
variation in RBC volume.
• Cannot be used as a diagnostictool.
• Does not indicate the nature of thedisease.