Test Background Information

The hematocrit test measures the percentage of red blood cells (RBCs) in a blood sample relative to its total volume. Because RBCs are responsible for transporting oxygen throughout the body, the hematocrit level can indicate how effectively RBCs can deliver oxygen to cells. This is crucial for the production of ATP (adenosine triphosphate), the energy storage molecule in the body.

To determine the percentage of red blood cells (RBCs) in blood, a sample is either centrifuged or left standing in an anticoagulant-coated (heparinized) glass tube. The components then separate into three distinct layers based on their density..

Red blood cells, the densest blood components, form a dark red column at the bottom of the tube. The volume of RBCs after centrifugation is also commonly referred to as packed cell volume (PCV). The PCV ranges from 41% to 50% in males and 36% to 44% in females.

White blood cells (WBCs; leukocytes) and platelets (thrombocytes) form a thin layer (1% of blood) directly above the RBCs. This layer is referred to as the buffy coat because of its light color.

The plasma, a pale, straw-colored fluid, fills the hematocrit tube above the buffy coat. It is composed mainly of water (92%) and transports a variety of dissolved and suspended substances. These substances include proteins, electrolytes, gases, and nutrients. The mean plasma percentage in males is ~49-58% and ~50-63% in females.

Blood Component Layers

Hematocrit levels that are too high or too low can indicate a blood disorder, dehydration, or other medical conditions.

Test Procedures

In a well-equipped lab, an automated (computerized) analyzer is often used to calculate the hematocrit. The analyzer determines the value by multiplying the red cell count by the mean cell volume (MCV). If an automated analyzer is not available, a ‘spun hematocrit’ can be performed. Using this method, the HCT can be measured by centrifuging the blood to separate the plasma from the RBCs and WBCs (white blood cells).

Step 1: Lance Finger

A lancing device is first loaded with a lancet. It is then adjusted so that the subject’s skin is penetrated to the proper depth. One of the subject’s fingers is selected, and the lancing device is positioned against the skin’s surface. The trigger button is pressed, and the skin is pierced. A small drop of blood will typically accumulate on the skin’s surface. If more blood is needed, squeeze the puncture site until the proper amount of blood appears.

Step 2: Draw Blood into a Hematocrit Tube

One end of a microhematocrit tube is inserted into the blood droplet. Capillary attraction will draw the blood up the narrow glass tube. When it is about 3/4 full, remove the tube. A lining of heparin, an anticoagulant, prevents the blood from clotting as it comes into contact with the tube.

Step 3: Seal the Hematocrit Tube with Clay

The blood end of the microhematocrit tube is inserted into a tray of sealing clay. The clay prevents the blood from draining out of the tube while it is being centrifuged.

Step 4: Place Hematocrit Tubes in Centrifuge

After being collected, the blood-filled microhematocrit tubes are carefully placed in a centrifuge. The tubes are positioned so that their sealed ends face out. A lid is placed over the tubes to secure their position while the centrifuge rotor spins.

Step 5: Set Spin Rate and Time on Centrifuge

The lid of the centrifuge is closed, and the apparatus is typically set to spin at 10,000 to 12,000 rpm for approximately 4 to 6 minutes.

Step 6: Spin Hematocrit Tubes

The rotor of the centrifuge is started. While the rotor is spinning, centrifugal force causes the blood in the tubes to separate into layers based on their density.

Step 7: Examine Blood Component Layers

Examine the hematocrit tubes to determine if there are three distinct layers, indicating complete separation and proper centrifugation.

Step 8: Determine Results Using a Hemocrit Reader Card

A hematocrit reader card is used to determine the percentage of RBCs in the blood sample. First, the glass tube is positioned on the left side of the reader card, with the bottom of the RBC column on the 0% line. Then, the glass tube is moved across the reader card until the top of the plasma column reaches the 100% line. The percentage of RBCs is then determined from the markings on the reader card.

Hematocrit Learning Activity

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References and Attributions

American Red Cross – “What Does Hematocrit Mean?”

OpenStax, Anatomy and Physiology: “An Overview of Blood.” (Access for free at https://openstax.org/books/anatomy-and-physiology/pages/1-introduction)

NIH – “Hematocrit.”

NIH – “Hemoglobin and Hematocrit.”

Wikipedia – “Hematocrit“