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 Packed Cell Volume (PCV)

Packed cell volume (PCV) is the volume occupied by the red cells when a sample of anticoagulated blood is centrifuged. It indicates relative proportion of red cells to plasma. PCV is also called as hematocrit or erythrocyte volume fraction. It is expressed either as a percentage of original volume of blood or as a decimal fraction.

USES OF PCV

• Detection of presence or absence of anaemia or polycythemia

• Estimation of red cell indices (mean cell volume and mean corpuscular haemoglobin concentration)

• Checking accuracy of haemoglobin value (Hemoglobin in grams/dl × 3 = PCV).

There are two methods for estimation of PCV: macro method (Wintrobe method) and micro method (micro hematocrit method). Micro method is preferred because it is rapid, convenient, requires only a small amount of blood, capillary blood from skin puncture can be used, and a large number of samples can be tested at one time. This method is also more accurate as plasma trapping in red cell column is less.

MACRO METHOD (WINTROBE METHOD)  OF PACKED CELL VOLUME (PCV)

Principle

Anticoagulated whole blood is centrifuged in a Wintrobe tube to completely pack the red cells. The volume of packed red cells is read directly from the tube.

An advantage with this method is that before performing PCV, test for erythrocyte sedimentation rate can be set up.

Equipment

1. Wintrobe tube: This tube is about 110 mm in length and has 100 markings, each at the interval of 1 mm. Internal diameter is 3 mm. It can hold about 3 ml of blood.

2. Pasteur pipette with a rubber bulb and a sufficient length of capillary to reach the bottom of the Wintrobe tube.

3. Centrifuge with a speed of 2300 g.

Specimen

Venous blood collected in EDTA (1.5 mg EDTA for 1 ml of blood) or in double oxalate. Test should be performed within 6 hours of collection.

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Method

1. Mix the anticoagulated blood sample thoroughly.

2. Draw the blood sample in a Pasteur pipette and introduce the pipette up to the bottom of the Wintrobe tube. Fill the tube from the bottom exactly up to the 100 mark. During filling, tip of the pipette is raised, but should remain under the rising meniscus to avoid foaming.

3. Centrifuge the sample at 2300 g for 30 min (To counterbalance a second Wintrobe tube filled with blood from another patient or water should be placed in the centrifuge).

4. Take the reading of the length of the column of red cells. Hematocrit can be expressed either as a percentage or as a fraction of the total volume of blood sample.

Significance

In anemia, PCV is below the lower level of normal range. PCV is raised in dehydration, shock, burns, and polycythemia.

After centrifugation of anticoagulated whole blood, three zones can be distinguished in the Wintrobe tube from above downwards- plasma, buffy coat layer (a small greyish layer of white cells and platelets, about 1 mm thick), and packed red cells. Normal plasma is straw-coloured. It is colourless in iron deficiency anaemia, pink in the presence of hemolysis or hemoglobinemia, and yellow if serum bilirubin is raised (jaundice). In hypertriglyceridemia, plasma appears milky. Increased thickness of buffy coat layer occurs if white cells or platelets are increased in number (e.g. in leukocytosis, thrombocytosis, or leukaemia). Smears can be made from the buffy coat layer for demonstration of lupus erythematosus (LE) cells, malaria parasites, or immature cells.

MICRO METHOD OF PACKED CELL VOLUME (PCV)

Principle

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.

Equipment

1. Microhematocrit centrifuge: It should provide relative centrifugal force of 12000 g for 5 minutes.

2. 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.

3. Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing.

4. Microhematocrit reader; if not available, a ruler or arithmetic graph paper.

Specimen

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.

Method

1. 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.

2. Seal the other end of the capillary tube (which was not in contact with blood) with a plastic sealant. If it is not available, heat-seal the tube using a spirit lamp.

3. 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.

4. Centrifuge at relative centrifugal force 12000 g for 5 minutes to completely pack the red cells.

5. 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.

6. 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: 

To obtain PCV, the above result is multiplied by 100.

GENERAL NOTES FOR PACKED CELL VOLUME (PCV)

1. Prolonged application of tourniquet during venepuncture causes hemoconcentration and rise in hematocrit.

2. Excess squeezing of the finger during skin puncture dilutes the sample with tissue fluid and lowers the hematocrit.

3. Correct proportion of blood with anticoagulant should be used. Excess EDTA causes shrinkage of red cells and falsely lowers the hematocrit.

4. Inadequate mixing of blood with anticoagulant, and inadequate mixing of blood before testing can cause false results.

5. Low hematocrit can result if there are clots in the sample.

6. Centrifugation at lower speed and for less time falsely increases PCV.

7. 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.

8. As PCV requires whole blood sample, it is affected by plasma volume (e.g. PCV is higher in dehydration, and lower in fluid overload).

9. 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.

10. 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

11. Automated hematocrit: In automated haematology analyzers, hematocrit is obtained by multiplying red cell count (in millions/cmm) by mean cell volume (in femtoliters).

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REFERENCE RANGES OF PACKED CELL VOLUME (PCV)

• 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%

CRITICAL VALUES  OF PACKED CELL VOLUME (PCV)

• Packed cell volume: < 20% or > 60%.