Lecture #5: The Hematocrit

  

Hematocrit (packed cell volume) of a sample is the ratio of the volume of erythrocytes to that of the whole blood. It is expressed as a percentage or preferably, as a decimal fraction. Before, it was often referred to as volume percent erythrocytes or volume of packed red cells.

The test measures the proportion of red blood cell to plasma in the peripheral blood, but not in the entire circulation. This is called then as the venous hematocrit. The ratio of the total erythrocyte mass to the total blood volume is the body hematocrit. Unless, otherwise specified, the term hematocrit often refers to venous hematocrit.

The volume of packed red cells is obtained by centrifugation of the blood sample (with an anticoagulant) for 30 minutes at 3,000 to 3,500 rpm.

In the process, spontaneous sedimentation is accelerated by centrifugation. The erythrocytes will be packed at the bottom of the tube and above them is a layer of leukocytes, platelets and nucleated erythrocytes called the buffy layer. Above the buffy coat is relatively cell–free plasma.

Clinical importance of hematocrit determination

1. Gives a rough estimate of the size of erythrocytes and the concentration of erythrocytes.

2. It is used in the calculation of mean corpuscular values and blood indices.

3. The buffy coat obtained from the hematocrit tube has numerous uses.

4. Hematocrit is a good simple screening test for anemia.

5. Since the inherent error obtained in hematocrit determination is lesser than erythrocyte count, it is not recommended to substitute hematocrit for erythrocytes count in routine hematological examination like complete blood count.

Normal values

            Men                 0.42 – 0.48

            Women           0.37 – 0.42

            Newborn         0.44 – 0.64

Methods of hematocrit determination

A.     Macromethods

1.      Wintrobe method

Anticoagulant used:          Double oxalate

Procedure:

a. Fill the Wintrobe tube with properly mixed blood with the use of Pasteur pipette.

b. Centrifuge for 30 minutes at 3,500 rpm.

c. Read the volume of packed cells at the right side calibration of the tube.

d. Calculate the hematocrit value from the following formula:

Hematocrit (EVF)        =          L₁         x          100

                                                L₂

L₁ – height of the red cell column in mm

L₂ – height of the whole blood column in mm

Note: Buffy coat is not included in L₁

2.      Haden’s modification method

Anticoagulant:       1.1% sodium oxalate

3.      Van Allen’s method

Anticoagulant:       1.6% sodium oxalate

4.      Sanford – Magath method

Anticoagulant:       1.3% sodium oxalate

5.      Bray’s method

Anticoagulant:       Heparin

            Note:

The principle employed in the last 4 methods is the same as that of Wintrobe method.

B.      Micromethods

1.      Adam’s micromethod

This method uses heparinized capillary hematocrit tube of about 7 cm long with an internal diameter of 1mm.

Procedure:

a. Fill the capillary tube with blood from a skin puncture (at least half full)

b. Seal the empty end in a small flame or plug with a modelling clay.

c. Place the tube in the radial groove of a microhematocrit centrifuge head with a sealed end away from the center.

d. Centrifuge for 5 minutes at 15,000 rcf.

e. The capillary tube is not calibrated and the hematocrit value is obtained by using commercially available ready device (graph)

C. Electrical method for measurement of packed cell volume

Principle:

Plasma is a conductor whereas red cells are resistors, so that a signal passed through a column of blood will be impeded to a degree proportional to the volume occupied by the red cells. This is the principle by which packed cell volume is estimated in some types of automated blood counting apparatus.

Advantages of micromethods over the macromethods:

1. Requires small amount of blood, so venipuncture may be avoided

2. Simplicity of the procedure

3. Low cost of apparatus

4. Free flowing blood is used

Disadvantages of micromethod:

1. Capillary blood is more variable than venous blood

2. The capillary tube is exclusively used for hematocrit and ESR reading cannot be possibly obtained

3. The buffy coat is not so distinct

Sources of error:

1. Speed and duration of centrifugation

2. Type and amount of anticoagulant

3. Excess anticoagulant causes shrinkage of cells and therefore decreases the hematocrit value.

4. Irregularity in the length and diameter of the tube

5. Errors in the sample; improper techniques in the collection of venous and capillary blood.

6. Failure to mix the blood properly before sampling

7. Leakage of blood in the micromethod

8. Errors in taking the reading and calculating the results.