ERYTHROCYTE SEDIMENTATION

RATE
COMPENTENCY BASED OBJECTIVES
➤ At the end of the exercise the student should be able to:
➤ 1. Enumerate the methods of performing ESR and correctly interpret the ndings
of performed ESR on a blood sample.
➤ 2. Enumerate the methods of performing PCV and correctly interpret the ndings
of performed PCV on a blood sample (Wintrobe’s tube).
➤ 3. Correctly calculate RBC indices from the given values of Hemoglobin, PCV and
RBC count.

fi
fi
BACKGROUND
➤ When anticoagulant mixed blood is allowed to stand undisturbed for a period of
time, the erythrocytes tend to settle (sediment) to the bottom. Two principal layers
are formed:
➤ an upper plasma layer
➤ a lower RBC layer.

➤ The rate of red cell sedimentation (estimated under standard conditions) is called
Erythrocyte Sedimentation Rate (ESR). It is expressed as mm in one hour.
STAGES
➤ Sedimentation takes place in the following 3 stages:
➤ Stage of rouleaux formation – it takes 10 minutes
➤ Stage of sedimentation – it takes 40 minutes
➤ Stage of packing of rouleaux – it takes 10 minutes
PROCEDURE
➤ It is done by the following methods:
➤ Wintrobe’s Method
➤ Westergren’s Method
➤ ESR Micromethod
➤ Automated ESR method
WINTROBE’S METHOD
➤ Wintrobe’s tube is a glass tube, closed at one end and open at the other. Its length is
11 cm and inner diameter is 2.5 mm. It has markings from ‘0 to 100’ on one side
and ‘100 to 0’ on the other side.
➤ Normal Values
➤ In Males: 0 to 9 mm in rst hour (FHr)
➤ In Females: 0 to 20 mm in rst hour (FHr)
➤ PROCEDURE: The Wintrobe’s tube is lled with anticoagulated (EDTA or Double
oxalate) blood up to 0 mark with the help of a Pasteur pipette, taking care to ensure
that no air bubbles form in the blood column. The tube is allowed to stand vertically
for 1 hour in a specially designed stand. After one hour the plasma column height is
noted in terms of mm after 1st hour (FHr).
fi
fi
fi
ADVANTAGES AND DISADVANTAGES
➤ Advantages:
➤ 1. Simple method, which requires only a small amount of blood and is safer for
blood handlers.
➤ 2. The anticoagulant used are solid hence no dilution of blood occurs.
➤ 3. PCV can also be done with the same sample and tube.

➤ Disadvantages:
➤ 1. It is not a sensitive index as the column of blood is short.
➤ 2. ESR of more than 100 mm cannot be measured.
WESTEGRENS METHOD
➤ Westergren’s pipette is open at both ends. It is 30 cm in length, with an internal diameter of 2.5 mm.
Its readings are 0–200 and is used for ESR determination only.
➤ Normal Value
➤ In Males: 3 to 5 mm in rst hour (FHr)
➤ In Females: 4 to 7 mm in rst hour (FHr)
➤ PROCEDURE:
➤ 1. 1.6 ml venous blood is mixed with 0.4 ml of 3.2% sodium citrate in a test tube (ratio 4:1). EDTA
blood can also be used but will need dilution either with citrate/saline before performing the test.
➤ 2. Now the tube is lled exactly up to zero mark with the help of rubber teat.
➤ 3. It is placed upright in a stand with a spring clip at the top and rubber stopper at the bottom.
➤ 4. The tube is allowed to stand for 1 hour.
➤ 5. The level of top of red cell column is read at the end of 1 hour.
fi
fi
fi
PRECAUTIONS
➤ The test should be performed as early as possible after the collection of blood, which
should be preferably a fasting sample.

➤ Care must be taken that there are no air bubbles while lling the tube with blood.

➤ The tube must be kept in a vibration-free bench in an absolutely vertical position.

fi
ESR MICROMETHOD
➤ Automated method with internal mixing and reading function.

➤ Minimal interference by lipids and bilirubin as it has an infrared sensor.

➤ Fast results (20 minutes).

➤ Results have good correlation with Westergren method.

➤ Very fast and accurate results.

➤ Can be done on EDTA blood.

➤ 2. Pathological conditions
➤ a. Tuberculosis
➤ b. Myocardial Infarction
➤ c. Anaemia
➤ d. Rheumatic fever
➤ e. Rheumatoid arthritis
➤ f. Macroglobulinaemia
➤ g. Multiple myeloma
➤ h. Temporal arteritis
➤ i. Hodgkin’s disease, where an ESR above 60 mm FHr indicates bad prognosis.
DECREASED ESR
➤ 1. Polycythaemia
➤ 2. Due to abnormal shapes of RBCs as in sickle cell anaemia or spherocytosis
➤ 3. Congestive Heart Failure
➤ 4. Hypo brinogenaemia
fi
PACKE CELL VOLUME
➤ Background
➤ Packed cell volume (Haematocrit) is the ratio of volume of packed red blood cells to
that of whole blood. It is expressed as percentage.
➤ Besides packed red blood cells and plasma, a bu y coat composed predominantly of
leucocytes is also seen at the junction of cells and plasma.
➤ NORMAL VALUES:
➤ 42 to 52% in adult males
➤ 36 to 47% in adult females
➤ PCV is estimated by two methods – Macro and Micro method.
ff
MACROMETHOD
➤ Material required:
➤ 1. Blood sample collected in Oxalate or EDTA anticoagulant.
➤ 2. Wintrobe’s haematocrit tube (Scale with markings from below upwards are used for PCV
determination).
➤ 3. Pasteur pipette.
➤ 4. Centrifuge machine.
➤ Procedure:
➤ 1. Shake the blood sample gently by rotating the vial on the tabletop manually.
➤ 2. Fill the Wintrobe’s tube using Pasteur pipette. Avoid trapping of air bubbles.
➤ 3. Place the tube in a centrifuge machine. Centrifuge at 3000 RPM for 30 minutes.
➤ 4. Note the lowermost height of column of packed red blood cells.
➤ Advantages
➤ 1. The same tube lled for ESR can measure PCV.
➤ 2. The colour of plasma gives indication of other conditions i.e. yellow in case of
jaundice, reddish due to haemolysis, milky due to lipemia and cloudy perhaps due to
multiple myeloma.
➤ Disadvantages
➤ 1. Carelessly cleaned or wet tubes give gross errors.
➤ Calculation:
➤ PCV = Height of RBC column/{Height of whole blood specimen X 100
fi
MICROMETHOD
➤ Background
➤ In the case of di culty in drawing su cient amount of blood, Micro hematocrit method is used. It is particularly useful in pediatric
patients. Blood is centrifuged in a sealed capillary tube and a special hematocrit scale reader is used to read PCV.
➤ Material required
➤ 1. Oxalated / EDTA blood or capillary blood.
➤ 2. Capillary hematocrit (microhematocrit) tubes – 75 mm in length with an internal diameter of 1 mm. May be heparinised if capillary
blood is to be used.
➤ 3. Soft wax, modeling clay or dental sealant to seal the end of the hematocrit tube.
➤ 4. MicroHematocrit reader.
➤ 5. MicroHematocrit centrifuge – runs at about 15,000 RPM.
➤ Procedure
➤ 1. Draw the specimen into a capillary hematocrit tube. Fill in the tube to about three-fourths of its length.
➤ 2. Seal both ends of the tube with soft wax, modeling clay or dental sealant, or ame.
➤ 3. Place the tube in Microhematocrit centrifuge. Centrifuge at 15,000 RPM for 5 minutes.
➤ 4. Note the reading against special hematocrit reader.
ffi
ffi
fl
➤ Advantages
➤ 1. Results are available within ve minutes.
➤ 2. More accurate measurement can be done and a smaller sample is required.
➤ Disadvantages
➤ 1. It requires a special centrifuge and disposable capillary tubes.
fi
➤ Increase in Hematocrit Values are Observed in:
➤ 1. Polycythemia
➤ 2. Hemoconcentration due to severe dehydration
➤ 3. Emphysema
➤ 4. Congenital heart disease
➤ 5. Burns and shock
➤ Decrease in Hematocrit Values are Observed in:
➤ 1. Anemia
➤ 2. Excessive uid volume of blood as in pregnancy (hydraemia of pregnancy)
fl
ABSOLUTE BLOOD CELL INDICES
➤ Absolute values are calculations based on measurable parameters in blood. They are very
important for diagnosis of hematological diseases as these values give quantitative
information about the red blood cell.
➤ Three indices are commonly calculated:
➤ 1. MCV (Mean Corpuscular Volume)
➤ 2. MCH (Mean Corpuscular Hemoglobin)
➤ 3. MCHC (Mean Corpuscular Hemoglobin Concentration)
➤ These are calculated by utilizing the following measured parameters.
➤ Hemoglobin concentration (Hb)
➤ Packed cell volume (PCV)
➤ Total RBC count (TRBC)
FORMULAE
➤ Determination of Mean Corpuscular Volume (MCV)
➤ MCV = PCV /Total RBCs in millions/cumm X 10
➤ Normal range = 82 to 92 cu microns ( )
➤ Determination of Mean Corpuscular Hemoglobin (MCH)
➤ MCH = Hb/RBC in million/cumm X 10
➤ Normal range = 27 to 32 pg
➤ Determination of Mean Corpuscular Hemoglobin Concentration (MCHC)
➤ MCHC = Hb/PCV X 100
➤ Normal range = 32–36%
fl
SIGNIFICANCE OF MCV
➤ MCV
➤ High MCV is found in:
➤ 1. Normal newborn
➤ 2. Reticulocytosis
➤ 3. Down’s syndrome
➤ 4. Liver disease
➤ 5. Hypothyroidism
➤ 6. Drug induced
➤ 7. Folate de ciency
➤ 8. Vit. B12 de ciency (Megaloblastic anemia)
➤ Low MCV is found in:
➤ 1. Iron de ciency anemia
➤ 2. Lead poisoning
➤ 3. Thalassemia
fi
fi
fi
SIGNIFICANCE OF MCH AND MCHC
➤ MCH
➤ High MCH is found in macrocytic anemia.
➤ Low MCH is found in hypochromic anemia.

➤ MCHC
➤ High MCHC is found in spherocytosis.
➤ Low MCHC is found in hypochromic anemia.
THANK YOU