When anti-coagulated blood is centrifuged at a standard speed, erythrocytes, which are heavier than white cells and plasma, will settle down at bottom. This red cells volume is known as Haematocrit or Packed Cell Volume (PCV).
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Haematocrit or PCV is the volume of red cells expressed as a percentage of whole blood.
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Methods:
There are two methods, used for the determination of haematocrit:
1. Macrohaematocrit
2. Microhaematocrit
Macrohaematocrit:
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A large volume of blood is required in this method. Approximately 2 to 4 ml is required.
Principle:
Anticoagulated blood is taken in a Wintrobe tube. Fill upto the uppermost mark and then rotate for desired length of time.
The packed cell volume (PCV) of red cells is directly read from the graduated tube as %.
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Requirement:
1. Blood specimen:
EDTA or double oxalated anti-coagulated blood is used in this method. Determine P.C.V. within six hr. of blood collection.
2. Wintrobe Tube:
It is 110 mm in length and 2.5 mm in diameter. The lower 100 mm are graduated or marked, from 100 at top and 0 (zero) at bottom for PCV.
3. Long necked pasture pipette or a special type of syringes is used for filling Wintrobe tube.
4. Centrifuge machine with known speed.
Procedure:
Mix 0.4 ml of EDTA with 2 ml blood. Fill the Wintrobe tube upto upper most mark with the help of pasture pipette or syringe. Fill the another Wintrobe tube to balance first one. If the blood sample is not available, fill the tube with water.
Place the Wintrobe tube in opposite side in centrifuge. Turn the centrifuge to slow speed, then slowly increase the speed to 3,000 rpm. Centrifuge for 30 min. at 3,000 rpm. After 30 min. switch off the centrifuge and allow it to stop by itself. Take out the Wintrobe tube and read PCV directly with the help of graduation mark given on the tube.
Normal Value:
i. In male – 42 to 50%
ii. In female – 36 to 38%
Microhaematocrit:
This method requires small amount of blood, 2 to 3 drops only. The blood can be obtained by finger puncture.
Principle:
Anti-coagulated blood is centrifuged in a sealed capillary tube, and then PCV is determined by a special haematocrit reader.
Requirement
1. Blood Specimen:
Blood from finger puncture may be used or EDTA or double oxalate venous blood can also be used.
2. Capillary Tube:
Use plain capillary tube for anti-coagulated venous blood and use heparinised capillary tube (Coated with heparin internally) for blood obtained from finger puncture. The capillary tube is approximately 75 mm in length.
3. Microhaematocrit Centrifuge:
This is a special type of centrifuge. It has speed about 15,000 r.p.m. The top of centrifuge is flat with grooves. The centrifuge also has timer, which is usually set for 5 min.
4. Haematocrit Reader:
There are several types of readers used for reading hct. The simplest method is use of card reader, which can be made by hand.
5. Clay:
This is used to seal the end of capillary tube.
Procedure:
Draw the blood sample into appropriate capillary tube with capillary action. Use plain tube for anti-coagulated blood and heparinised tube for plain blood. In case of finger puncture, the blood should flow freely with little pressure. Now wipe off the first drop and then collect the blood specimen.
Fill the tube about 3/4th length with blood. Seal the another end of the tube with clay or wax or ultimately by heating. The sealing should be about 2 mm deep. Place two hct tubes in the groove of centrifuge exactly opposite to each other.
It is not necessary that the capillary tube have exact amount of blood level. In case, if there is no filled capillary tube to balance we can use an empty capillary tube. Centrifuge at 13,000 ± 2000 rpm.
Remove capillary tube from centrifuge. It will show three layers. Top layer is of plasma or serum; the middle layer is thin creamy white in colour and is known as Buffy coat. It is a layer of WBC; the last layer is the column of RBC. Use the hct reader for finding the value of hct.
Card Reader:
The reader is used as, hold the tube against Scale so that the bottom of red cell is matched with 0 (zero) line of the card. Move the tube across the card until the uppermost line of plasma is matched to 100% line of card.
Check to make sure that bottom of red cell column is still in the line of zero and the tube should be straight and vertical. The line that passes through the top of the column of RBC gives the hct. Value.
Importance:
Decrease in PCV is suitable measure for anaemia. The fall in hct may be seen in decreased oxygen supply to cells, heart disease or malignant condition, hct also rise in case of dehydration.