When anticoagulated blood is allowed to stand vertically. RBC’s settle towards the bottom of the tube under the influence of gravity resulting in packed column in a given interval of time leaving the clear plasma above. The process of sedimentation is called ESR.
Why do cells settle
Density of RBC’s is greater than that of plasma
RBC’s tend to aggregate to form rouleaux. Rouleaux are piled up RBC’s that become heavier & sediment faster.
Mechanism of ESR
Normal RBC’s settle slowly as they do not form rouleaux due to the negative charge on their surface which causes them to repel each other
Plasma proteins especially fibrinogen adhere to the red cells membranes & neutralize the surface negative charges promoting cell adherence & rouleaux formation
ESR is directly proportional to the weight of the cell aggregate and inversely proportional to the surface area.
Stages of ESR
Stage of rouleaux formation/aggregation or lag phase
Where piling up of RBC’s take place -10 -15 minutes
Stage of sedimentation/settling or decantation phase
This is actual phase of falling of RBC’s – 40 minutes
Stage of packing or Stationary phase -10min
Packing of sedimented RBC’s occurs in column due to over crowding – 10minutes
What is Zeta potential
Zeta potential results from negatively charged sialic acid groups on red cell membrane.
Factors affecting ESR
How does the plasma factors affect ESR
Fibrinogen, globulin & cholesterol – Increases ESR by decreasing negative charge on RBC’s
Plasma albumin – decreases ESR
Lecithin – decreases ESR
How does the number, size and shape of RBC’s affect ESR
Number
Increased in red cell mass –decrease ESR
Decreased Red cell mass – Increase ESR
Size:
Microcyte –sediment slowly.
Macrocyte-sediment rapidly from normocytes
Shape:
Sickle cell –decreased ESR because abnormal cell decreases rouleaux formation
Splerocytes –sediment slowly
Why ESR is raised in anemia
Sedimentation rate is directly proportional to weight of the cell aggregates and inversely proportional to the surface area. Microcytes have decreased surface area to volume ratio
How does the Rouleaux formation affect ESR
When RBC aggregate to form rouleaux, density increases & they settle down. The area is much less than sum of the area of constituent corpuscle. Hence it is important factor that increases ESR
How does the age and sex affects ESR
Age: In infants ESR is low. As the age increases it gradually increases and in elderly people ESR is higher
Sex: Women has slightly higher ESR than male because of their low PCV
What are the changes in ESR during pregnancy
ESR increases from about the 3rd month of pregnancy and returns back to normal after 4 weeks of delivery
How does the temperature affects ESR
Higher temperature causes false high results due to reduction in plasma viscosity
Refrigerated blood should be brought to normal room temperature
Rise in every 3oc will cause 1mm rise in ESR
What is the best anticoagulant for ESR estimation
K2EDTA or 3.8% Trisodium citrate
Heparin should not be used as it alters the cell membrane potential
What are the tube factors which affect the ESR
ESR is higher in longer tube
Inner diameter should be 2.5mm or more to overcome capillary attraction
Position of the tube:
Tube should be perfectly vertical
Angle of 3o from vertical can increase ESR by 30%
What are the other factors affecting ESR
Sunlight – Direct sunlight on tube increases ESR
Vibration – Reduces ESR
Hemolysed or clotted sample – ESR is less as clot traps fibrinogen and hence there is no rouleaux formation
Time – ESR should be estimated with in two hours of sample collection. If there is delay then the sample should be stored at4oC and test should be performed with in 6 hours.
Drugs –
Drugs like OCPs, Theophylline, Dextran, Methyldopa, Pencillamine, and Vit A increases ESR
Aspirin, Cortisone and Quinine decreases ESR
Conditions with increased ESR
Pregnancy
Multiple myeloma
Anaemia
Inflammatory disease
Macrocytosis
Acute & chronic infection
Rheumatic fever
Rheumatic arthritis
Tuberculosis
SLE
Conditions with decreased ESR
Hyperviscosity
Decreased fibrinogen levels
Polycythemia
Sickle cell anaemia
Spherocytosis
Microcytosis
Factors that decrease ESR
Extreme leukocytosis
Technical factors like improper dilution, inadequate mixing
Clotting of blood samples
Short ESR tube
Vibration during testing
Clinical significance of ESR
ESR is non specific hematological test for inflammation caused by infections, malignant conditions, degenerative and autoimmune diseases. ESR is raised in these conditions which is associated with increase in plasma proteins particularly fibrinogen, immunoglobulins and C-reactive protein
It is a prognostic test
It detects the presence and severity of disease
It gives an idea about the activity of disease
Repeated ESR estimation helps in prognosis & follow up of disease
Different methods for ESR estimation
Wintrobes method
Westergren’s methos
Micro sedimentation method (Landau method)
Automated ESR method
Wintrobe method:
Wintorbes tubes –
closed at one end
Length 11cm
Internal bore of the tube -2.5 cm
Tube is calibrated both sides 0-10 & 10-0
Can hold 0.7 to 1ml of blood
Sample –EDTA sample
Procedure must be performed within 2hrs of blood collection
Procedure –
Mix anticoagulated blood
Fill the wintrobe tubes by Pasteur pipette upto mark ‘0’
Place the tube vertically in stand
Note ESR at the end of 1hr
Normal values –
Male -0-10mm in 1st hr
Female -0-20mm in 1st hr
Advantages –
Small amount of blood is required.
PCV can be done by the same tube.
Filling of tube by Pasteur pippete eliminates chances of infection
Disavantages –
Because of short column the result may be inappropriate.
ESR of more than 100mm cannot be measured
Westergreen method
Sample – 3.8% trisodium citrated anticoagulant blood. 6ml of blood is mixed in 0.4ml of Trisodium citrate (ratio of blood to TSc -4:1)
Westergren tube :
Length -30cm
Internal bore diameter -2.5mm
Open at both ends
Calibarated from top to bottom -0-200
can hold – 2ml of blood
Procedure:
Mix the blood thoroughly
Draw blood into the tube upto mark ‘0’ with the help of rubber bulb.
Wipe out blood from the bottom of tube
Transfer the tube to westergren stand & stand it vertically.
Height of clear plasma on the top of the tube is measured after 1 & 2 hrs
Normal values
Male – 0.5 mm in 1st hr; 6-10 mm in 2nd hr
Female -8-10 mm in 1st hr; 16-20 mm in 2nd hr
Advantages – More reliable and gives accurate results as sedimentation of RBC’s occur better in longer tube.
Disadvantages:
More blood is required
Difficult & fill the blood in tube
PCV cannot the done
Mouth pipette may be hazardous
Microsedimentation method (Landau method)
Used in infants when blood is insufficient for the above methods.
Capillary blood can be used
Equipments
Landau pipette with stand: Similar to RBC pippete with markings 0-50mm
5.0 gm/dL sodium citrate is used as an anticoagulant
Landaus apparatus consists of a metal stand with 6 or 12 capillary tubes that may be supplemented with a syringe. Capillary tube has a length of 12 cms. Its inside diametre is 1mm. Little ampoule is present in the upper part of the tube. Lower part is provided by two markings, 12.5 and 62.5mm respectively from the tip.
Procedure
capillary tube is fitted with syringe. Draw 5% sodium citrate up to the lower mark on the stem and then blood up to the second mark from the incision in the finger tip, heel or ear lobe. Wipe off excess blood on the tip of the pipette. Draw both the solutions in the bulb. Set the upper level of the mixture to 0 mark at the top. Detatch the suction device, and then place the pipette in a vertical position on the stand. Note the reading at the end of the 1st hour.
Automated methods
Ves-matic 20
ESR STAT –plus
SEDIMAT
Zeta sedimentation ratio (ZSR)
Ves-matic 20
Measures 20 blood samples at a time
results will be given in 22 min .
Results are comparable to Westergren method
Procedure:
Blood is collected in special cuvette & sample is mixed . sample is allowed to stand at 180 slant from vertical. Opto electrical sensor measures ESR. Data are elaborated and printed.
ESR STAT PLUS:
It is Centrifugation based method
Provides result in 5 min
Working principle: Sample is placed in centrifuge. Infrared laser tracks the erythrocyte plasma interface & takes multiple measurements. Linear portion of sedimentation is identified. software algorithm determines ESR result.
SEDIMAT
Filled sediplast westergren pippet is placed into SEDIMAT automated ESR reader which accelerates sedimentation under controlled conditions
The reader displasys the results after 15 mins.
Zeta sedimentation ratio
ZSR is performed using a special small base capillary tube that is filled with blood & spun for 3 to 4 minutes in a special centrifuge called Zetafuge.
Centrifuge alternately compacts & disperses the RBC’s under standardised centrifugal force.
RBC’s are subjected to alternate force of compaction & dispersion within a vertically held tube. Extent of compaction is inversely proportional to the repulsive force between the red cells namely zeta potential.
This procedure is unaffected by either sex or by anemia and responds in a linear manner to increase in fibrinogen gamma globulin
Blood sample of 100µl contained within vertically oriented capillary tube is subjected to 4 cycles of alternate dispersion & compaction. Tube is spun at the speed of 400 rpm. Each cycle should last for 45 seconds
At the conclusion of this process, the hematocrit of the blood in the red cell containing portion of the capillary tube is measured on a special reader to obtain a value called Zetacrit.
A measure of the closeness which red cells will approach each other under a standardised stress is ZSR
ZSR is expressed in new units % rather than mm/hr. Normal range is – 40% -51%
It is rapid, correct for anemia & require small blood sample which is desirable for pediatric patients
Advantages of automated methods
Provides safety as sample manipulation is required
Interface with laboratory information system
Use smaller sample volumes
Provide more rapid results
Source of error
Improper ratio of blood anticoagulant
Hemolysed sample or Clotted blood
Presence of air bubbles
Error due to sunlight vibration small bore size, dirty & wet tube
References
Praful B. Godkar, Darshan P. Godkar.Textbook of medical laboratory technology 2007. Second Edition
Sabitri sanyal, Aparna Bhattacharya.Clinical pathology A practical manual 2017. Third edition.
Brian S. Bull, J.Douglas Brailsford. The Zeta Sedimentation Ratio. Blood 1972;vol 40(4):550-559
Gunner Nielsen and Inge Rode-Moller. Heparin blood and Landau’s microsedimentation method. Acta Medica Scandinavia 1943;Vol.CXIII,Fasc V:https://doi.org/10.1111/j.0954-6820.1943.tb09177.x
By
Dr.Shyam Sundara Rao (Professor of Pathology, Narayana Medical College, Nellore)
Dr. Bhavana Grandhi (Associate Professor of Pathology, Narayana Medical College, Nellore)
Dr. V.Shanthi (Professor of Pathology, Narayana Medical College, Nellore)
