Red blood cells, also known as erythrocytes and commonly abbreviated as RBC in routine laboratory tests, are the most numerous type of blood cell in the blood. They serve as the primary medium for oxygen transport in the blood of vertebrates and also possess immune functions. Mature red blood cells in mammals are anucleate, meaning they lack DNA. They also have no mitochondria and produce energy through glucose metabolism. In addition to transporting oxygen, they carry a portion of carbon dioxide—appearing dark purple when transporting carbon dioxide and bright red when transporting oxygen—and they also have immune functions.
Principle of Action of Red Blood Cell Diluent (RBC Dilution), An isotonic diluent is used to dilute blood at a specific ratio. The diluted blood is then loaded into a hemocytometer, and the number of red blood cells in a defined volume is counted under a microscope. The red blood cell concentration per liter of blood is subsequently calculated via conversion. This RBC diluent is for research use only and not intended for clinical diagnosis.
Self-Prepared Materials:
1. Fresh whole blood or EDTA-anticoagulated whole blood
2. Micropipettes
3. Hemocytometers
4. Microscopes
Operating Procedures (For Reference Only):
1. Take a medium-sized test tube and add 2 mL of RBC Dilution to it.
2. Use a clean, dry micropipette to draw 10 μL of peripheral blood or anticoagulated blood. Wipe off excess blood from the outside of the pipette, insert the pipette to the bottom of the RBC Dilution, then gently aspirate the supernatant to rinse the pipette 2–3 times and mix the solution thoroughly immediately.
3. Use a clean micropipette to load the red blood cell suspension into the counting chamber of the hemocytometer, taking care to avoid bubble formation or overflow. Let it stand at room temperature for 1–3 minutes.
4. Place the hemocytometer under the high-power microscope and count the red blood cells in five medium squares in total—the four corner medium squares and the central medium square within the large central counting square—one by one. For cells overlapping the grid lines, follow the counting principle of "count cells touching the top and left lines, not the bottom and right lines".
Calculation:
RBC count/L= Number of red blood cells in 5 medium squares×5×10×200×10⁶=Number of red blood cells in 5 medium squares×10¹⁰
×5
| Convert the count from 5 medium squares to 1 large square
|
×10
| Convert the volume of 1 large square (0.1 μL) to 1.0 μL
|
×200
| The actual blood dilution ratio is 201-fold, calculated as 200-fold for convenience
|
×10⁶
| Convert from 1 μL to 1 L
|
Precautions:
1. Do not over-squeeze the puncture site during blood collection, and ensure an appropriate needle puncture depth.
2. Do not convert hemoglobin concentration to red blood cell count.
3. Within the reference range, the difference between two replicate red blood cell counts should not exceed 5%.
4. For your safety and health, wear a lab coat and disposable gloves during all operations.
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