To perform ESR
Definition:
The erythrocyte sedimentation rate (ESR) measures the rate of settling (sedimentation) of erythrocytes in
anticoagulated whole blood. Anticoagulated blood is allowed to stand in a glass tube for 1 hour and the
length of column of plasma above the red cells is measured in millimeters; this corresponds to ESR.
STAGES OF ERYTHROCYTE SEDIMENTATION RATE
There are three stages of erythrocyte sedimentation:
• Stage 1: Formation of rouleaux or lag phase (10 minutes): Red cells stack together like a
pack of coins.
The ESR depends mainly on this stage.
• Stage 2: Sinking of rouleaux or decantation phase (40 minutes): Rapid and constant
sedimentation. The longer the tube, the longer is this stage and higher
the value of ESR.
• Stage 3: Packing of rouleaux (10 minutes): Slow
sedimentation.
INDICATIONS FOR MEASUREMENT OF ERYTHROCYTE SEDIMENTATION RATE
It is recommended to measure ESR (i) when infectious, inflammatory, or a neoplastic disease is suspected in
symptomatic individuals in whom a specific diagnosis has not been established
(ii) to monitor disease activity in tuberculosis, inflammatory arthritis, rheumatic fever, Hodgkin’s disease, giant
cell arteritis, and polymyalgia rheumatica, and
(iii) as a diagnostic criterion for temporal arteritis and polymyalgia rheumatica.
ESR can also be helpful in distinguishing iron deficiency anemia from anemia of chronic disease in patients
known to have inflammatory disease.
METHODS FOR ESTIMATION OF ERYTHROCYTE SEDIMENTATION RATE
These include:
• Westergren method
• Wintrobe method
• Zeta sedimentation ratio
• Micro-ESR
Westergren Method
International Council for Standardization in Hematology recommends Westergren method for
estimation of ESR.
Equipment and Reagent
1. Westergren ESR tube: This is a straight glass pipette measuring 300 mm in length and
calibrated in mm from 0-200 (top to bottom). The markings are only over the lower 2/3rds of the
tube. The tube is open- ended. Internal diameter should not be less than 2.55 mm. The tube
should be clean and dry.
2. Westergren stand: This holds the tube in a motionless, vertical position
3. Anticoagulant-diluent solution: Trisodium citrate dihydrate is the anticoagulant of choice. Its
composition is as follows:
Trisodium citrate dihydrate 32.08 gm Distilled water upto 1000 ml
It is filtered through a sterile membrane (0.22 μm) into a sterile container and stored in a refrigerator
at 4°C. It keeps for several months, but if it becomes turbid (due to the growth of moulds), it should
be discarded.
Alternate anticoagulant is EDTA (1.5 mg/ml); however, such anticoagulated blood sample should be
diluted with trisodium citrate just before the test (1 volume of trisodium citrate to 4 volumes of blood).
Specimen: Venous blood is collected in trisodium citrate solution in 4:1 (blood: citrate) proportion. If
specimen is kept at room temperature, test should be carried out within 4 hours of blood collection; if stored
at 4°C, a delay of up to 6 hours is permissible. Blood anticoagulated with EDTA can be tested within 24
hours if stored at 4°C (provided it is diluted with trisodium citrate before testing).
Method
1. Mix anticoagulated blood sample thoroughly. The Westergren tube is filled with the blood sample up to the
“0” mark. A rubber bulb or a mechanical device should be used for filling. There should be no air bubbles in
the blood.
2. The tube is placed in a strictly vertical position in the ESR stand and left undisturbed for 1 hour. It should
not be kept in direct sunlight and should not be subjected to vibrations.
3. After exactly 1 hour, read the height of the column of plasma above the red cell column in mm.
4. Express the result as:
Erythrocyte sedimentation rate = ——— mm in 1 hour.
Precautions
1. Use the correct proportion of blood and anticoagulant. Mix blood and anticoagulant thoroughly. There
should be no clots and air bubbles in blood.
2. The reference range relates to test performed at room temperature of 18-25°C. If temperature is higher,
ESR will increase and different reference range will have to be derived.
3. ESR tube must be kept vertically. Even a slight tilting will increase the ESR.
Other Methods
Wintrobe Method
Wintrobe tube can be used for estimation of both ESR and packed cell volume (PCV). After obtaining
ESR in the first hour, the tube can be spun in a centrifuge to get PCV. Wintrobe’s method is more reliable
when ESR is low, while Westergren’s method is more sensitive for high ESR. EDTA or double oxalate is
used as an anticoagulant. Length of Wintrobe tube is shorter (110 mm) and internal diameter is about 3
mm.
Zeta Sedimentation Ratio (ZSR)
A special device called as zetafuge is required. ZSR is not affected by anemia, unlike Westergren
method.
Micro-ESR
This method uses capillary blood that is collected in anticoagulant-coated capillary tubes. It is more
suitable in small children.