When anticoagulated blood is allowed to stand undisturbed condition for a period of time, the erythrocytes tends to sink to the bottom. Two layers are formed, the upper plasma layer and lower sedimented erythrocyte layer. The rate at which the red cells fall is knows as Erythrocyte Sedimentation Rate (ESR).
Mechanism of sedimentation of RBCs :
The ESR is consist of mainly four phases. These are aggregation, rouleaux formation, sedimentation and packing.
1st 15 minutes : Phase of minimum fall : The red cells suspended in a column of citrated blood undergo rouleaux formation in the plasma and become heavier. Sedimentation in this phase is very low.
2nd 15 minutes : Phase of moderate falling : Fibrinogen and globulin in the plasma develop fine threads and build up a network. The rouleaux of the red cells get tapped in the mash of network and become heaviest. It starts to settle quickly.
3rd 15 minutes : Phase of maximum fall : In this phase the rate of fall of the protein network with red cell mass is maximums.
4th 15 minutes : Phase of packing : In this phase the sedimented red blood cells and protein mass undergoes packing to the bottom.
Factors influencing ESR :
- Plasma : RBCs carry a negative electronic charge, where as plasma carries a positive charge. Any conditions in plasma that increases it positive charge; increase rouleaux formation and increases ESR by lengthing stage-I (aggregation and rouleaux formation.). Fibrinogen, globulin and cholesterol accelerate while albumin retards sedimentation. Hence, ESR is increase in any conditions that increases fibrinogen (tissue break-down as in infection and tuberculosis) or globulin (rheumatic fever, multiple myaloma and kala-azar).
- Red Blood Cells : Increase in blood cell counts as in Polycythemia retards sedimentation due to the jostling (or pushing one another) effect on the cells. Low blood cell count in anaemia tends to increase ESR. However, altered shaped of the RBCs as in Sickle cell anaemia and microcytic hypochromic anaemia tends to prevent rouleaux formation and decrease ESR. In Sickle cell anaemia the red cell resist to rouleaux formation that’s why ESR is decreased
- Physiological Variation : ESR is low in infants, increase upto puberty, and then decreases upto old age when aging it increased. ESR is greater in women then men. It increases after the third month of pregnancy and returns to normal by about third or fourth weeks after delivery (postpartum).
- Temperature of the environment : ESR is increases when temperature of the environment is increase. It is directly proportional to the temperature.
- Difference between specific gravity of Red blood cell and plasma.
- Diameter and length of the ESR tube.
- Position of the ESR tube : Normally ESR tube is placed vertical position (90°). If the tube is placed slightly slant, ESR is increases. For every 3° of the tube position ESR is increase approximately 30 mm.
Clinical Significance :
- Changes in ESR are not diagnostic for any specific disease.
- ESR has prognostic value. Elevated ESR (as in tuberculosis, rheumatoid fever, multiple myaloma, kala-azar etc.), if returns to normal suggests improvement in clinical course.
- ESR decrease in the following conditions :
- Increase albumin level in the blood.
- Congestive cardiac failure.
- States of sever dehydration like cholera, gastroenteritis.
- Sickle cell anaemia.
- Hypochromic microcytic anaemia.
- Infants and old age.
- ESR increase in the following conditions :ESR increase in all condition where there is tissue breakdown or where there is entry of foreign protein in the body.
- Rheumatic fever.
- Myocardial infarction.
- Rheumatoid arthritis.
- Systemic Lupus Erythromatous.
- Chronic Osteomyelitis.
- Chronic lungs abscess.
- Multiple myelomatosis. - In case of Multiple Myelomatosis approximately 130 – 140 mm/ 1st hour of ESR is increase.
Determination of Erythrocyte Sedimentation Rate (ESR)
Determination of ESR is done by several methods. These are as follows:
In the developing countries former two methods are used for ESR determination in the laboratory.
Wintrobe’s tube :
- It is 110 mm long tube with a 3.0 mm internal bore. It is graduated from ‘0’ upto ‘100’ mm (10 cm). The scale with the descending order is used for ESR determination.
- It is also used for Packed Cell determination.
Westergren’s tube :
- It is 300 mm long tube with a 2.5 mm internal bore. It is graduated from ‘0’ upto ‘200’ mm (20 cm). The scale with descending order is used for ESR determination.
- It is more specific then the Wintrobe’s method and others methods.
Normal value :
By Westergren’s method :
- Male : 0-15 mm/1st hour
- Female : : 0-20 mm/ 1st hour
By Wintrobe’s method :
- Male : 0-9 mm/1st hour
- Female : 0-20 mm/ 1st hour
Principle of the Test :
When anticoagulated blood is allowed to stand undisturbed condition for a period of time, the erythrocytes tends to sink to the bottom. Two layers are formed, the upper plasma layer and lower sedimented erythrocyte layer. The rate at which the red cells fall is knows as Erythrocyte Sedimentation Rate (ESR) and give the result as mm/1st hour.
DETERMINATION OF ERYTHROCYTE SEDIMENTATION RATE BY WESTERGREN METHOD
Patient should be fasting for 12 to 16 hours. Collect venous blood. 2.0 ml of blood is collected in a vial containing 0.5 ml of 3.8% Sodium citrate anticoagulant. Fasting EDTA anticoagulated blood is also used for ESR determination.
- Westergren’s tube with stand.
- Test tube with test tube rack.
- 3.8 % Sodium Citrate solution.
- Stop watch.
- Blood drawer.
- Blood drawer attached with clean and dry Westergren’s tube.
- Drawn 3.8% sodium citrate upto ‘150’ mark from ‘200’ mark in a test tube.
- Drawn well mixed anticoagulated blood upto ‘0’ mark from ‘200’ mark of the tube and mixed with 3.8% sodium citrate present in the test tube.
- Mixed well, filled the Westergren tube exactly upto ‘0’ mark (avoid air babbles formation in the tube).
- Placed the tube upright position in the ESR stand, it should fit evenly into the groove of the stand and it allowed standing undisturbed condition exactly for 1 hour. Set the time in Stop-watch.
- Exactly after 1 hour, note the level to which the red cell column has fallen.
- Report the result in terms of mm/1st hour.
DETERMINATION OF ERYTHROCYTE SEDIMENTATION RATE BY WINTROBE'S METHOD
Fresh fasting (12 to 16 hours fasting) EDTA anticoagulated and undiluted blood.
- Wintrobe’s tube with stand.
- Special Pasteur pipette attached with rubber tied.
- Stop watch.
- Mixed the blood carefully in the vial.
- Filled the Wintrobe's tube to the ‘0’ mark by using a special Pasteur pipette.
- Placed the tube in exact vertical position in the stand.
- Set the Stop-watch for 1 hour.
- At the end of one hour note the level of erythrocyte column and give the result in terms of mm after first one hour (mm/ 1st hour).