MICRO METHOD FOR ESTIMATION OF PACKED CELL VOLUME (PCV) OR HEMATOCRIT
Anticoagulated whole blood is centrifuged in a capillary tube of uniform bore to pack the red cells. Centrifugation is done in a special microhematocrit centrifuge till packing of red cells is as complete as possible. The reading (length of packed red cells and total length of the column) is taken using a microhematocrit reader, a ruler, or arithmetic graph paper.
- Microhematocrit centrifuge: It should provide relative centrifugal force of 12000 g for 5 minutes.
- Capillary hematocrit tubes: These are disposable glass tubes 75 mm in length and 1 mm in internal diameter. They are of two types: plain (containing no anticoagulant) and heparinised (coated with a dried film of 2 units of heparin). For plain tubes, anticoagulated venous blood is needed. Heparinised tubes are used for blood obtained from skin puncture.
- Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing.
- Microhematocrit reader; if not available, a ruler or arithmetic graph paper.
Venous blood collected in EDTA (dipotassium salt) for plain tubes or blood from skin puncture collected directly in heparinised tubes. Venous blood should be collected with minimal stasis to avoid hemoconcentration and false rise in PCV.
- Fill the capillary tube by applying its tip to the blood (either from skin puncture or anticoagulated venous blood, depending on the type of tube used). About 2/3rds to 3/4ths length of the capillary tube should be filled with blood.
- Seal the other end of the capillary tube (which was not in contact with blood) with a plastic sealant. If it is not available, heatseal the tube using a spirit lamp.
- The filled tubes are placed in the radial grooves of the centrifuge with the sealed ends toward the outer rim gasket. Counterbalance by placing the tubes in the grooves opposite to each other.
- Centrifuge at relative centrifu-gal force 12000 g for 5 minutes to completely pack the red cells.
- Immediately remove the tubes from the centrifuge and stand them upright. The tube will show three layers from top to bottom: column of plasma, thin layer of buffy coat, and column of red cells.
- With the microhematocrit reader, hematocrit is directly read from the scale. If hematocrit reader is not available, the tube is held against a ruler and the hematocrit is obtained by the following formula:
Length of total column in mm
To obtain PCV, the above result is multiplied by 100.
- Prolonged application of tourniquet during venepuncture causes hemoconcentration and rise in hematocrit.
- Excess squeezing of the finger during skin puncture dilutes the sample with tissue fluid and lowers the hematocrit.
- Correct proportion of blood with anticoagulant should be used. Excess EDTA causes shrinkage of red cells and falsely lowers the hematocrit.
- Inadequate mixing of blood with anticoagulant, and inadequate mixing of blood before testing can cause false results.
- Low hematocrit can result if there are clots in the sample.
- Centrifugation at lower speed and for less time falsely increases PCV.
- A small amount of plasma is trapped in the lower part of the red cell column which is usually insignificant. Increased amount of plasma is trapped in microcytosis, macrocytosis, spherocytosis, and sickle cell anemia, which cause an artifactual rise in hematocrit. Larger volume of plasma is trapped in Wintrobe tube than in capillary tube.
- As PCV requires whole blood sample, it is affected by plasma volume (e.g. PCV is higher in dehydration, and lower in fluid overload).
- Expression of PCV: Occasionally, PCV is expressed as a percentage. In SI units, PCV is expressed as a volume fraction. Conversion factor from conventional to SI units is 0.1 and from SI to conventional units is 100.
- Rules of 3 and 9: These rules of thumb are commonly used to check the accuracy of results and are applicable only if red cells are of normal size and shape.
• Hemoglobin (gm/dl) × 3 = PCV
• Red cell count (million/cmm) × 9 = PCV
- Automated hematocrit: In automated hematology analyzers, hematocrit is obtained by multiplying red cell count (in millions/cmm) by mean cell volume (in femtoliters).
- Adult males: 40-50%
- Adult females (nonpregnant): 38 45%
- Adult females (pregnant): 36-42%
- Children 6 to 12 years: 37-46%
- Children 6 months to 6 years: 36 42%
- Infants 2 to 6 months: 32-42%
- Newborns: 44-60%
- Packed cell volume: < 20% or > 60%